CN101600727A - Viral inhibitory nucleotide sequences and vaccine - Google Patents

Abstract

The present invention relates to inhibitory nucleotide signal sequence or " INS " sequence in the lentiviral gene group.Particularly, the present invention relates to be present in AGG motif in all viral genome.The AGG motif can be for example by reducing or keep the low steady state levels of viral RNA in host cell, and induce and/or keep virus hide virus is had retarding effect.In one aspect, the invention provides the vaccine that comprises viral nucleic acid or produce, in described viral nucleic acid, the AGG sequence is suddenlyd change from viral nucleic acid.In yet another aspect, the invention provides the method and composition of the function that is used for influencing the AGG motif and the method that is used for other INS sequences of identifying virus genome.

Description

Viral inhibitory nucleotide sequences and vaccine

[001] all publications of mentioning herein, patent application, patent and other bibliographys are integrated with this paper in full by reference with it.

The present invention finishes under the government by USDOE project U.S.DE-FG02-90ER40542 supports.Therefore, United States Government has some right of the present invention.

Background of invention

[002] lentivirus is in the retrovirus family of virus.Term " lenti " is the Latin of " slowly ".Slow virus is characterised in that to have long latency and direct infection adjacent cells and needn't form the extracellular particle.Their turnover at a slow speed (turnover) adds their long-term abilities that keeps in cell, makes slow virus be good at escaping infected experimenter's immune response especially.Slow virus comprises immunodeficiency virus, for example human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and equine infectious anaemia virus (equineinfectious anemia viruses, EIAV).Slow virus infection can cause serious disease, and, if do not treat, can be fatal.In recent years, several antiretroviral drugs and the medicinal mixture that reduce virus load and improve the symptom of HIV infection have been developed.Yet although they are successful, these medicines can not be eradicated virus infection usually entirely.On the contrary, virus remains among the infected experimenter to hide usually.Also carried out for example effort of the vaccine of HIV of the anti-slow virus infection of many generations.Yet, up to now, also do not have the vaccine of commercially available acquisition.Therefore, need develop anti-slow virus for example newtype drug and the vaccine of HIV in this area.

Summary of the invention

[003] the invention provides and compare the trinucleotide sequence motifs AGG that (over-represented) in the genome of HIV virus, excessively occur with the gene that contrasts in the people's gene group.The AGG motif also is present in other viral genomes with high level.It is believed that the AGG motif is inhibitory nucleotide signal sequence or and " INS " sequence.

[004] in one embodiment, thus the present invention relates to carry out the viral nucleic acid that sudden change changes over one or more AGG sequences non-AGG sequence.In some embodiments, viral nucleic acid is from HIV virus.In other embodiments, thus having carried out the viral nucleic acid that sudden change changes over non-AGG sequence with one or more AGG sequences is present in gag, pol or the env gene.

[005] in another embodiment, the present invention relates to produce the method for the viral nucleic acid with one or more AGG that suddenlyd change, this method comprises to be provided the viral nucleic acid that comprises one or more AGG sequences and one or more AGG sequences is changed over non-AGG sequence.The AGG sequence can be positioned at or derive from virus genomic any position, comprises coding and non-coding region.In another embodiment, if the AGG sequence is positioned at the zone of the viral nucleic acid of coded protein, the non-AGG sequence of selecting to be changed over by the AGG sequence is so that it can influence sequence, structure, function or immunogenicity by described viral nucleic acid encoded protein matter sharply.In other embodiments, viral nucleic acid is a HIV nucleic acid.

[006] on the other hand, thus the present invention relates to have has carried out the genomic mutated viruses that sudden change changes over one or more AGG sequences non-AGG sequence.In some embodiments, mutated viruses is the HIV virus of sudden change.

[007] in another embodiment, thus the present invention relates to not to virus but comprise having carried out the recombinant virus that sudden change changes over one or more AGG sequences the nucleic acid sequence of non-AGG sequence.In another embodiment, mutated viruses nucleic acid is sudden change HIV nucleic acid.

[008] in other embodiments, the present invention relates to from the virus protein of mutated viruses nucleotide sequence expression, thereby described mutated viruses nucleotide sequence has carried out sudden change one or more AGG sequences are changed over non-AGG sequence.In another embodiment, thus the present invention relates to one or more AGG sequences be changed over the HIV protein that the sudden change HIV nucleotide sequence of non-AGG sequence is expressed from having carried out sudden change.

[009] in another embodiment, thus the present invention relates to comprise has carried out the virus vaccines that sudden change changes over one or more AGG sequences the nucleic acid sequence of non-AGG sequence.In another embodiment, the present invention relates to the HIV vaccine, having carried out suddenlys change changes over one or more AGG sequences the HIV nucleotide sequence of non-AGG sequence thereby described HIV vaccine comprises.

[010] in another embodiment, the present invention relates to virus vaccines, described vaccine comprises any above-mentioned nucleic acid sequence, comprising the nucleic acid sequence that has than the AGG motif of finding in the nucleotide sequence of natural viral still less.In another embodiment, the present invention relates to the HIV vaccine, described HIV vaccine comprises the HIV nucleotide sequence that has than the AGG motif of finding in the nucleotide sequence that is in corresponding natural HIV strain still less

[011] in another embodiment, the present invention relates to can be than the corresponding wild C-type virus C virus vaccines of highland marking protein more, and wherein said virus vaccines comprises the nucleotide sequence that has than wild-type virus nucleotide sequence AGG sequence still less.In another embodiment, the present invention relates to can be than the corresponding wild type HIV virus HIV vaccine of the ability of highland marking protein more, and wherein said HIV vaccine comprises the nucleotide sequence that has than wild-type HIV nucleic acid sequence AGG sequence still less.

[012] in another embodiment, the present invention relates to comprise the virus vaccines that produces from viral nucleic acid, one or more AGG sequences are changed over non-AGG sequence thereby described viral nucleic acid has carried out sudden change.In another embodiment, the present invention relates to comprise the proteinic HIV vaccine that produces from HI V nucleic acid, one or more AGG sequences are changed over non-AGG sequence thereby described HIV nucleic acid has carried out sudden change.

[013] in another embodiment, the present invention relates to comprise as by vaccine provided by the invention be selected from the composition of other components of pharmaceutically acceptable thinner, carrier, vehicle and adjuvant.

[014] in another embodiment, the present invention relates to be used for immune experimenter with antiviral method, described method comprises the vaccine of the present invention of the experimenter being used significant quantity.In one embodiment, the present invention relates to be used for immune experimenter with antiviral method, described method comprises the virus of the experimenter being used significant quantity, thus described virus carried out the sudden change one or more AGG sequences are changed over non-AGG sequence.In another embodiment, the present invention relates to be used for the method for immune experimenter with anti-HIV, described method comprises the nucleic acid of the experimenter being used the coding virus protein of significant quantity, thereby described nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.In another embodiment, in another embodiment, the present invention relates to be used for immune experimenter with antiviral method, described method comprises the virus protein from the viral nucleic acid generation of the experimenter being used significant quantity, thereby described viral nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.In some embodiments, the present invention relates to be used for the method for immune experimenter with anti-HIV.

[015] in another embodiment, the present invention relates to be used for the method for indentifying substance, described reagent suppresses or stimulates the proteinic generation of generation, slow virus of viral RNA or the generation of virion, or inhibition or viral the hiding of stimulation.In another embodiment, described method comprises the subject cell (described nucleic acid sequence comprises at least one AGG motif that is mutated into non-AGG sequence) that the control cells (described nucleic acid sequence comprises at least one AGG motif) that comprises at least a nucleic acid sequence is provided and comprises at least a nucleic acid sequence, described subject cell is contacted with one or more reagent with described control cells, with at least a reagent of evaluation, with compare in control cells, described reagent suppresses or stimulates the generation of viral RNA in subject cell, the generation of proteinic generation of slow virus or virion, or inhibition or viral the hiding of stimulation.In other embodiments, described reagent suppresses or stimulates the proteinic generation of generation, HIV or generation of HIV particulate or the inhibition of HIV RNA or stimulate HIV to hide.

[016] in another embodiment, the present invention relates to be used to identify the method for AGG motif wedding agent.In another embodiment, method comprises the nucleic acid that tried that the contrast nucleic acid that comprises at least one AGG motif is provided and comprises at least one AGG motif that has been mutated into non-AGG sequence, the described nucleic acid that tried is contacted with one or more reagent with described contrast nucleic acid, with identify at least a reagent, described reagent in conjunction with described contrast nucleic acid but debond described tried nucleic acid or with than its with tried the nucleic acid bonded more high-affinity combine and contrast nucleic acid.

[017] in another embodiment, the present invention relates to AGG motif wedding agent, for example use the AGG motif wedding agent of an evaluation in the method for the present invention.

[018] in another embodiment, the present invention relates to suppress or stimulate the AGG wedding agent with the nucleic acid bonded reagent that comprises at least one AGG motif be used to identify this type of compositions and methods.

[019] the invention still further relates to the method for the INS sequence of gene except the AGG motif that is used for identifying virus.

[020] these and other embodiments of the present invention are further described in appended specification sheets, accompanying drawing and the claim.

Summary of drawings

[021] Fig. 1, (a) and (b) part shows the nucleotide sequence of HIV-1gag gene and the aminoacid sequence of encoded protein matter.(a) part shows nucleotide site 1 to 780, and (b) part shows nucleotide site 781 to 1543.This sequence comprises 44 AGG motifs.On nucleotide sequence, be illustrated in some sudden changes in the producible possible sudden change in these AGG motifs.For example, described figure shows that the AGG motif that starts from nucleotide site 1442 can change over AAG from AGG.Fig. 1 illustrates 38 AGG sudden changes.

[022] Fig. 2 shows the result who expresses from Gag in instantaneous 293 cells of transcribing of 4 independent transfection experiments.Two different Gag sequences are that codon optimized form (Adarc) and the motif that we produce optimized form (RK).The RK form of Gag gene has than the about 2 times of high expression of described codon optimized form.

[023] Fig. 3 shows the immunogenicity of Gag dna vaccination in the mouse.Two kinds of multi-form Gag are prepared to dna vaccination, and are injected into the Balb/C mouse, strengthen in the 4th week then.On the 4th week and the 6th time-of-week point, measure anti-Gag antibody horizontal by ELISA.Our form RK-Gag is in 5 times of high immune responses of the codon optimized form of the 4th all induction ratios, and this increases to 6 times difference after the 6th week.

Detailed Description Of The Invention

[024] the present invention relates in the viral genome the inhibitory nucleotide burst or " INS " sequence. INS sequence of the present invention can be for example thin the host by reducing viral RNA Level among the born of the same parents or make it keep low steady state levels and virus is had the inhibition effect. This The INS sequence of invention can also play an important role in that virus lays dormant is interim. In one aspect, The invention provides the INS sequence that comprises wherein carried out sudden change viral nucleic acid vaccine or from The vaccine that described viral nucleic acid produces. In yet another aspect, the invention provides for affecting INS The method and composition of the function of sequence and for the identification of the method for INS sequence. This paper describes These and other embodiments.

[025] Definition

[026] unless content is clearly pointed out other meanings, singulative " ", " one " " be somebody's turn to do " and comprise plural connotation. Therefore, for example, " virus " comprises a plurality of these viroids.

[027] term " AGG ", " AGG motif ", " AGG sequence " and " AGG order The row motif " be used interchangeably in this article, have three nucleosides of sequence A GG in the expression nucleic acid The continuous sequence of acid.

[028] term used herein " mutant " refers to by one or more nucleosides Modified nucleic acid or protein that sour or amino acid whose insertion, disappearance and/or displacement change. Example As, the term mutant for example is used in reference to and passes through with one in another nucleotide subsitution AGG motif Individual or a plurality of nucleotides, or insert one or more nucleotides with destruction AGG motif, or disappearance One or more nucleotides in the AGG motif and they are not replaced into other nucleotides and carry out Change is to destroy the nucleic acid of AGG motif. When specification and claim are mentioned the AGG motif prominent Become non-AGG sequence, this comprises with one in another nucleotide subsitution AGG motif or many Individual nucleotides, or insert one or more nucleotides with destruction AGG motif, or lack from AGG One or more nucleotides and they are not replaced into other nucleotides. .

[029] term used herein " wild type " or " WT " refer to not be changed with broken The nucleic acid of bad AGG motif and the virus, carrier and the cell that comprise described nucleic acid. Term is " wild Type " also refer to the protein by this class nucleic acid coding. Therefore, term " wild type " comprises natural Nucleic acid, virus, carrier, cell and protein. Yet, in addition, term " wild type " Also comprise non-natural nucleic acid, virus, cell and protein. For example, unless otherwise noted, The nucleic acid, virus, carrier and the cell that have carried out hereditary change are included in term " wild type " In,, these nucleic acid, virus and cell destroy therein AGG as long as not changing.

[030] as used herein, term " homologue " is and the nuclear of mentioning herein Nucleotide sequence total at least about 60%, about 70%, about 80%, about 90% or higher same The nucleotide sequence of one property, the wild type slow virus nucleotide sequence of for example mentioning herein. Hundred Proportion by subtraction homogeneity can be any number (comprising 60% and 99.9%) in 60% to 99.9% the scope.

[031] term " with coming from " also is used in reference to the protein (example that has and mention herein Such as the slow virus protein of mentioning herein) amino acid sequence total at least about 60%, 70%, 80%, 90% or the protein of the amino acid sequence of higher homogeneity. Percentage homogeneity can be Any number in 60% to 99.9% the scope (comprising 60% and 99.9%). In some enforcement sides In the case, the homologue of the protein of describing herein has with the wild type of describing slowly sick herein Poisonous protein is similar structure and/or function and/or immunogenicity basically.

[032] as used herein, " virus " comprises the RNA that has around genetic stocks Or any infectious particles of the protein coat of DNA core. As used herein, term " virus " also refers to all strains, separated strain and the clade of all DNA and RNA virus (clade). Virus includes but not limited to all adenovirus, Alfamovirus, verdant disease Poison genus, different light Tobamovirus, Alphacryptovirus, β fat hair bacteriophage, Alphanodoavirus, α Papillomavirus (Alphapapillomavirus), Alpharetrovirus, α virus Genus, A Liushen mink Tobamovirus, grapevine leafroll virus genus, Hostis, aquatic couple of RNA Tobamovirus, Aquareovirus, arenavirus genus, Arterivirus, ascovirus Genus, African swine fever virus genus, gland thymus gland Tobamovirus, gold Pueraria lobota Tobamovirus, fowl Astrovirus, Oat Tobamovirus, Aviadenovirus, Avibirnavirus, Avihepadnavirus, fowl Poxvirus, avian paramyxovirus belong to (Avulavirus), banana top bundle Tobamovirus, shaft-like DNA Tobamovirus, Barnavirus, spiral shell vibrios minute phage, Begomovirus, Betacryptovirus, β fat hair bacteriophage, Betanodovirus, β Papillomavirus, β Epsilonretrovirus ε, Betatetravirus, bocavirus belong to, ripple is received Tobamovirus, Bracovirus, Short neck Densovirus, bromovirus, bymovirus, capillovirus Genus, Capripoxvirus, cardiovirus, Carlavirus, Carmovirus, Caulimovirus, Carmovirus (Cavemovirus), Chlamydia are small Bacteriophage, Chlorovirus, Chloriridovirus genus, chrysovirus, Circovirus, Linear Tobamovirus, coccoliths Tobamovirus, colorado tick fever Tobamovirus belong to (Coltivirus), Comovirus, coronavirus genus, corticovirus genus, cricket Fiber crops illnesss Tobamovirus, pumpkin mosaic virus genus, BCTV genus, cypovirus, Cystovirus genus, cytomegalovirus genus, Cytorhabdovirus, Dianthovirus (Dainthovirus), δ Papillomavirus, Deltaretrovirus, Densovirus, Dependovirus, ebola virus genus, Peas lug mosaic virus genus, enterovirus genus, elder brother Worm biplate section RNA Tobamovirus, A Entomopoxvirus, B Entomopoxvirus, C insect acne disease Poison genus, Ephemerovirus, ε Papillomavirus, ε retroviruse belong to, equine rhinoviruses Genus, equine rhinoviruses genus, Erythrovirus, Δ Papillomavirus, broad bean wilt virus belong to, Fijivirus genus, Flavivirus, depression Tobamovirus, small spindle bacteriophage, γ fat hair are bitten Thalline (Gammalipothrixvirus), γ Papillomavirus, Gammaretrovirus, merchant Flagellate virus genus, Granulovirus, little shape Tobamovirus (Guttavirus), ring disease Poison belongs to (Gyrovirus), Hantavirus, Hemivirus, prosperous Nipah virus genus, hepatitis Poison genus, hepadnavirus genus, hepatovirus, Hypovirus, Ichnovirus, Channel-catfish fish Herpesvirus, reovirus genus, isometrical variable Tobamovirus, ILTV Genus, Influenzavirus A, Influenzavirus B, influenza orthomyxovirus belong to C type, thread Tobamovirus, ι Papillomavirus (Iotapapillomavirus), sweet potato viruses belong to, iris Tobamovirus, Infectious salmon anaemia virus belong to, repeat Tobamovirus, κ Papillomavirus (Kappapapillomavirus), ridge Tobamovirus, Lagovirus, λ Papillomavirus (Lambdapapillomavirus), Leishmaniavirus, lentivirus, hare acne disease Poison belongs to, levivirus genus, Luteovirus, the latent Tobamovirus of lymph, Lymphocystivirus, Lyssavirus, maize chlorotic mottle virus belong to (Machlomovirus), orange mulberry mosaic disease Poison genus, Grapevine fleck virus genus, mammal Astrovirus, fork mandarin orange (oranges and tangerines) Tobamovirus (Mandarivirus), Marafivirus, Marburg virus genus, horse Garrick Tobamovirus, Marnavirus, mastadenovirus, Mastrevirus, enlargement are thin Cellular virus genus, Metapneumovirus, Metavirus, piconavirus belong to, mitovirus Genus, Molluscipoxvirus, Morbillivirus, Mupapillomavirus, MCMV Genus, fly reovirus genus (Mycoreovirus), Na Yiluo Tobamovirus, nanometer Tobamovirus (Nanovirus), Narnavirus, Necrovirus, Nepovirus, Nova Tobamovirus, grain outer Rhabdovirus, nucleopolyhedrosis virus genus, Nucleorhabdovirus, μ Papillomavirus (Nupapillomavirus), helmet Tobamovirus, bromovirus, Omegatetravirus, occasion type Papillomavirus, Orbivirus, positive Bunyavirus (Orthobunyavirus), positive liver nuclifort Tobamovirus, orthopoxvirus, just exhale The lonely Tobamovirus of intestines, Oryzavirus (Oryzavirus), Pa Nike Tobamovirus (Panicovirus), Parapoxvirus genus, the lonely Tobamovirus of secondary intestines, partitivirus belong to (Patitivirus), parvovirus belongs to Group, Peroplaneta fluligginosa Densovirus, plague virus belong to, morning glory leaf vein transparent sample Tobamovirus (Petuvirus), brown algal virus genus, Phlebovirus, Phytoreovirus, π nipple Tumor virus genus, plasmavirus genus, Plectrovi, Pneumovirus, Polerovirus, Polyomavirus, potexvirus, Potyvirus, parasitic phycovirus belong to, gold Phycovirus genus, pseudovirus genus, Ranavirus, needle phycovirus genus, Respirovirus, monkey Tobamovirus (Rhadinovirus), Rhinovirus, Roseolovirus, rotavirus, wind Exanthema virus genus, Rubulavirus, ancient bacteriophage, ryegrass mosaic virus belong to, bed ripples virus Genus, Seadornavirus, association Tobamovirus (Sequivirus), sialidase Tobamovirus, Simplexvirus, mottle virus genus, conveyor screw minute phage, Spumavirus, pig Tobamovirus, θ Papillomavirus in Poxvirus, tectivirus genus, the victory (Thetapapillomavirus), the high native Tobamovirus of holder, Tombusvirus, tomato puppet Curly top poison genus, Orbivirus, Tospovirus, totivirus genus, Trichovirus, Triticum Tobamovirus, tungro bacilliform virus genus, Tymovirus, Varicellavirus, water Bubble property Tobamovirus, vesicular rass Tobamovirus, Grapevine virus belong to, stunt virus belongs to, white spot virus belongs to, ξ Papillomavirus (Xipapillomavirus), Yatapoxvirus or ζ papillomatosis Poison belongs to (Zetapapillomavirus) or its any combination.

[033] term " retrovirus " as used herein refers to all reverse transcription diseases The poison all strains, separated strain and clade, include but not limited to all α retrovirus, β retrovirus, δ retrovirus, ε reverse transcription disease, γ retrovirus, foam disease Poison and slow virus.

[034] term " slow virus " as used herein refers to all of all slow virus Strain, separated strain and clade include but not limited to bovine immunodeficiency virus, equine infectious poor Mass formed by blood stasis virus (EIAV), feline immunodeficiency virus (FIV), Caprine arthritis encephalitis virus, myelin Virus, human immunodeficiency virus I type (HIV-1), human immunodeficiency virus come off-are short of breath II type (HIV-2) and simian immunodeficiency virus (SIV).

[035] as used herein " HIV " in the term refers to HIV-1 and HIV-2 All strains, separated strain and clade. Therefore, unless otherwise noted, when using term HIV And not during specified type (namely not indicating 1 type or 2 types), suppose that HIV-1 and HIV-2 refer to, All strains, separated strain and the clade that comprise HIV-1 and HIV-2.

[036] as used herein term " protein " and " peptide " refer to amino acid whose The polymer chain. Although term " peptide " is generally used for referring to the amino acid whose polymer chain of relatively lacking, And term " protein " is used in reference to longer amino acid whose polymer chain, but just can be taken as egg The molecule of white matter exists some overlapping with the molecule that can be taken as peptide. Therefore, term " egg White matter " and " peptide " in this article can Alternate, and when using this type of term, it Be not intended to limit by any way the length of the amino acid whose polymer chain of mentioning. Unless in addition Outer proposition, term " protein " and " peptide " should be interpreted as comprising the specified protein of mentioning All fragments, derivative, variant, homologue and analogies, and can comprise natural amino Acid or synthetic amino acid.

[037] term " corn " and " immunogenic composition " are used interchangeably in this article, Refer to can the inducing anti-disease poison immunoreactive reagent or composition. In another embodiment In, the invention provides and can induce anti-slow virus for example HIV-1, HIV-2, SIV, FIV Or the immunoreactive vaccine of EIAV. Term " vaccine " and " immunogenic composition " comprise Preventative or preventive vaccine and therapeutic vaccine. Vaccine combination of the present invention also can be with multiple Different viral cross reactions and anti-described virus effectively. For example, immunogenicity of the present invention Composition can lack with the immunity of virus, slow virus and/or the number of different types of number of different types Fall into venereal disease poison cross reaction and effectively resist described virus. Similarly, immunogenicity of the present invention Composition can cross reaction between the different strains of identical virus and clade. For example, have Also can effectively resisting according to immunogenic composition of the present invention of the strain of the anti-HIV in effect ground A plurality of strains of HIV.

[038] term " protein vaccine ", " proteinaceous as used herein Vaccine " and " subunit vaccine " be used interchangeably, refer to comprise the protein of slow virus or virus The vaccine of component.

[039] term used herein " reagent " generally is used for representing any molecule, for example Protein, peptide or medicine include but not limited to reagent, inhibition AGG base in conjunction with the AGG motif The another kind of examination of the reagent of the function of order, the reagent that stimulates the function of AGG motif, inhibition or stimulation With the reagent of the combination of AGG motif, comprise the nucleic acid of the AGG motif with sudden change or by described The vaccine of nucleic acid preparation, the molecule used altogether with vaccine of the present invention etc.

[040] term " host " refer to can by virus, slow-virus infection or can be used for the training Any vaccine strain, virus, carrier, plasmid or the egg of supporting, increasing or expressing description herein Any animal of white matter or cell type (comprise zooblast, bacterial cell, yeast cells and Insect cell).

[041] " experimenter " used herein refers to and can use according to epidemic disease of the present invention it Any animal of seedling or reagent comprises people and other mammalian species.

[042] " immunogenicity " comprises the immunoreactive ability of material incentive.Immunogenicity is that the existence of specific antibody is measured by determining for described material for example.The existence of antibody by the method known in the art for example the ELISA assay method detect.

[043] in one aspect, the present invention relates to have the virus of number of the AGG sequence of minimizing.In some embodiments, the present invention relates to slow virus.In other embodiments, the present invention relates to slow virus HIV.

[044] HIV biology

[045] HIV genome encoding some protein, some of them protein are generated as " polyprotein (poly-protein) " that produces the difference in functionality entity in proteolysis cutting back.All protein by the HIV genome encoding (including but not limited to " polyprotein " and their proteolysis cleaved products) and can be described as " HIV albumen ", " HIV peptide ", " HIV polyprotein ", " protein of the present invention ", " polyprotein of the present invention " or " peptide of the present invention " within the scope of the invention in this article.INS of the present invention can be present in the proteinic nucleotide sequence of coding any or all these.INS of the present invention also can be present in the genomic non-coding region of HIV.

[046] for example, the HIV genome encoding can be cut into the proteic pr55GAG albumen of p17MA, p24CA, p7 and p6 of the core of forming virus by the virus protein enzyme.The Pr160GAG-POL precursor protein produces the polysaccharase polyprotein that produces and be cut into subsequently reversed transcriptive enzyme (RT), RNA enzyme H, proteolytic enzyme (PR) and intergrase (IN) by translation frameshift (translatiomal frame shifting).The Gp160 envelope protein is cut into by leukoprotease virus is attached to the Gp120 albumen (ENV) of CD-4 acceptor and co-receptor and peplos is merged Gp41 transmembrane protein into the thin pleura of host.These albumen are main structural protein of HIV particulate and enzyme.Proviral the transcribing (Tat) that two kinds of other albumen Tat and Rev regulation and control are integrated and the mRNA (GAG-POL and virogene RNA) of not montage are from extremely cytoplasmic transhipment (Rev) of nucleus.The expression and the time-event of virogene regulated and control in these activities in replicative cycle.Four kinds of accessory protein: Vpu, Vif, Vpr and Nef are the joints (adaptor) that form mixture with cell protein, and it strengthens the generation of infectious virus in vivo, but has littler phenotype in some cell cultures.All these HIV protein within the scope of the present invention.

[047] HIV virus is attached to T cell and monocyte, the CD-4 albumen and the co-receptor of described gp120ENV protein binding cell surface by using its gp120ENV albumen.T cell expressing cytokine receptor CXCR4, monocytes CCR5 co-receptor, peripheral blood lymphocyte is expressed both.The proteic hereditary change generation of ENV is had a liking for monocyte virus (R5 virus), is had a liking for T cell virus (X4 virus) and anphotropc virus (dual tropic viruse).Co-receptor adheres to for ENV that to merge with Gp41 be essential.The almost completely anti-HIV of people that lacks normal HIV co-receptor (for example because they have CCR5/ δ 32 polymorphisms) infects.(referring to, for example, people such as Deng, Nature (1996) the 381st volume, people such as p 661-6 and Samson, Nature, (1996) the 382nd volume p722-5).The medicine of blocking-up CCR5 co-receptor now carries out in the clinical trial.

[048] after HIV virus and cytogamy, the nucleoid particle copies as the DNA copy by using reversed transcriptive enzyme (RT) with viral RNA, and particle moves to nucleus, at this its use intergrase (IN) viral DNA is integrated into cellular genome.It is essential many transcription factor binding site points that long terminal repetition (LTR) dna sequence dna comprises for producing virus mRNA from the DNA that integrates.Except the TATA element, in LTR, there are two NF κ b sites and three Sp-1 sites by cell transcription factor identification.Also there is site in addition at LEF, ETS and USF transcription factor.These cell transcription factors help initial transcribing, but this takes place with low-down level.After producing TAT, it is in conjunction with cell protein (cyclin T1) and TAT-cycT1 mixture, then in conjunction with the RNA ring structure (being called TAR) in the virus mRNA.The TAT-cycT1 mixture is then in conjunction with the CDK9 protein kinase, the carboxy terminal of a subunit of its phosphorylation rna plymerase ii.This is that viral RNA is transcribed the effective initial necessary of (it increases by 100 times).Produce the different viral RNA molecule of kind more than 30 by these incidents.They are divided three classes: (1) is used to produce the RNA of GAG, POL and complete virus genomic not montage, (2) be used to produce the RNA of local montage that the proteic size of ENV, Vif, Vpu and Vpr is about 5.0Kb and the RNA (1.7-2.0Kb) of the little montage that (3) translate into REV, TAT and Nef.The mRNA for complete montage to nucleus is the most effective with these rna transports.Therefore, early stage after infection, have only TAT, REV and NEF to be produced effectively.TAT increases by 100 times of transcription rates with combining of TAR then.Bigger, montage or not fully the mRNA of montage only REV albumen produce and in conjunction with the Rev response element in the ENV gene (Rev-responsive element, RRE) after, could more effectively transport into tenuigenin.Like this, the arrangement of time (timing) of the viral life cycle of synthetic regulation and control of TAT and REV.

[049] except TAT, in the HIV genome, there is the second cover signal of the steady state levels that reduces viral RNA in the cell.These signals are called inhibitory nucleotide signal sequence (INS sequence).In the genomic gag/pol of HIV zone, identified before supposition the zone that comprises INS (referring to people such as Schneider, Journal of Virology, (1997), the 71st volume, p.4892-4903).In the previous research of being undertaken by people such as Schneider, remove the AUUUA pentanucleotide and reduce AU content and do not change the coding capacity in zone thereby the zone of the INS sequence that comprises supposition suddenlyd change.Have been found that these sudden changes cause the level of HIV RNA to increase as many as 70-130 doubly.At the INS series jump with under the situation that functional r EV exists, the increase of the HIV RNA in the cell of infection is than high 160 times under these two kinds of non-existent situations of difference in functionality.

It is believed that [050] HIV virus obtains favourable aspect by the low steady state levels with viral RNA.Propose, the viral Billy of quick copy and quick cell killing uses virus generation every cell virus number still less in slower cycle.In fact, as viewed for for example poliovirus, virus produces and cell kills fast and effectively, causes virus immunity removing completely usually and to the immunity with postoperative infection.On the contrary, be retained in the cell with the virus in cycle of prolonging and can escape or reduce their effect of immunity system opposing at least.

[051], do not identify the sequence (people such as Schneider) of NIS before although the sudden change in the zone that comprises INS of the supposition of gag/pol gene adds up.The zone that comprises INS is used for reducing the steady state levels of the cell viral RNA of infection.There are at least three kinds of possible mechanism, comprise the reduction of (1) transcription rate, the increase of the RNA degradation rate of (2) HIV RNA, (3) HIV RNA is to the reduction of extranuclear transport velocity people such as () Schneider.

[052] the insufficient immune response to Env and Gag is for the subject matter in the effort of the vaccine of HIV-1 in generation.About dna vaccination, immune response is relevant with expression level, so the increase of the expression of these ORF can alleviate the significant obstacle to the structure of great-hearted vaccine.Infer because the protein binding site in groups of encoding among their RNA to have processing problems and can not transport from nucleus effectively from the big mRNA of HIV.Identifying and remove the signal that causes nuclear restriction (nuclear confinement) can increase the expression level of these ORF significantly and improve immune response.

[053] genome of human immunodeficiency virus's 1 type (HIV-1) comprises 9 open reading frames (ORF), and all open reading frames are by selecting montage from single promoter expression.The splicing form of these 6 ORF of Gag, Pol, Env, Vpu, Vif and Vpr and full length mRNA are included in the REV response element (RRE) of encoding among their RNA together.Under the non-existent situation of REV albumen, these 6 ORF express not enough.Other 3 ORF, Tat, Rev and Nef do not rely on REV albumen and express effectively.

[054] as if the mRNA that comprises RRE also comprises the signal of undetermined prevention normal expression or signal in groups.It is the nuclear restriction that these ORF express insufficient major cause.Compare with the known encoding gene in groups in the people's gene group, the genome of HIV-1 has unusual Nucleotide and distributes.The percentage ratio of the average VITAMIN B4 in the clinical separation strain of HIV-1 is higher than average man's encoding gene.Be widely used in the optimized strain of codon system in this experiment and the vaccine test and can increase by 500 to 1000 times of expression levels that are transfected into the Gag in people's cell.Yet the remarkable increase of the expression that is caused by the codon optimization can solve the problem that nuclear is isolated (nuclearisolation) at most indirectly.

[055] assembling of HIV virion occurs on the cytolemma of GAG-POL polyprotein packaging virus RNA wherein.Virion sprouts from the plasma membrane that has now comprised HIV ENV and the proteinic host cell of gp41.After virion discharges, virus protein enzyme (PR) cutting GAG-POL polyprotein, thus produce maturation and infectious particles.It is believed that Vif participates in the assembling of virus.In addition, NEF and Vpu participate in the proteic degraded of cell CD-4 and from the film releasing virus of the cell that infects.As if Vpr albumen be integrated into virus particle and working in early days of infecting, particle transported into nucleus in assembling, and the antiviral cell enzyme activity of Vif antagonism in cell.The infectious virus that discharges is attached to and infects other cells, and the lymphocytic progressive infection of CD-4 causes immunity system to lose ability with killing.

[056] INS sequence of the present invention

[057] because genetic code is a degeneracy, thus nucleotide sequence can be on nucleotide level different mutually but encode identical protein or peptide.Yet, in the common selection pressure that exists concrete codon selection and AT/GC content of occurring in nature.Also exist selection pressure by amino acid whose frequency in the nucleotide sequence coded protein and order (order).Developed each this type of selection pressure stdn, calculated the average frequency of the sequence motifs (for example, length is the sequence motifs of 2 to 8 Nucleotide) of expecting in the genome then and the actual frequency of this frequency and these motifs method relatively.This method is described in common unsettled temporary patent application number 60/808,420, also is described in people (2005) such as Robins, Journal of Bacteriology, and the 187th volume, p.8370-74, its content is integrated with this paper by reference.This method can be used for producing a series of insufficient oligonucleotide sequence motifs that excessively occur and/or occur in genome.This type of insufficient sequence excessively occurs and/or occur can comprise function information.

[058] in the present invention, people's such as Robins method be used for seeking HIV-1 and people's gene group this type of insufficient sequence motifs excessively appears and occurs.Deficiency excessively appears and occurs in discovery sequence motifs AGG in reciprocity mutually people's gene in the HIV genome.For example, Fig. 1 is presented at 48 AGG motifs identifying in the HIV-1gag gene.The AGG motif of identifying in the gag gene above 2/3rds is not present in the reading frame of coded amino acid, and this hints that these sequences are owing to the reason of selecting is not guarded on amino acid/protein level.Yet the variation of the 3rd codon position is common in different HIV strain isolateds, also finds the AGG motif even also is conservative especially in the 3rd position of codon.In addition, find that the AGG motif is in more than 400 HIV-1 strain system that has analyzed and guard in HIV-2 and other slow viruss (comprising FIV, SIV and EIAV).

[059] INS sequence of the present invention is for example in the genome of may be able to the be present in many dissimilar viruses of AGG sequence motifs.In one embodiment; the present invention relates to the INS sequence in the genome of any virus family; described virus includes but not limited to adenovirus; Alfamovirus; Allexivirus; different light Tobamovirus; Alphacryptovirus; β fat hair phage; Alphanodoavirus; α Papillomavirus (Alphapapillomavirus); Alpharetrovirus; Alphavirus; A Liushen mink Tobamovirus; grapevine leafroll virus belongs to; Hostis; Aquabirnavirus; Aquareovirus; arenavirus genus; Arterivirus; Ascovirus; African swine fever virus belongs to; gland thymus gland Tobamovirus; gold Pueraria lobota Tobamovirus; the fowl Astrovirus; the oat Tobamovirus; Aviadenovirus; Avibirnavirus; Avihepadnavirus; Avipoxvirus; avian paramyxovirus belongs to (Avulavirus); banana top bundle Tobamovirus; Badnavirus; Barnavirus; spiral shell vibrios minute phage; bean golden mosaic virus belongs to; Betacryptovirus; β fat hair phage; Betanodovirus; the β Papillomavirus; Betaretrovirus; Betatetravirus; bocavirus belongs to; ripple is received Tobamovirus; Bracovirus; short neck Densovirus; bromovirus; bymovirus; capillovirus belongs to; Capripoxvirus; cardiovirus; Carlavirus; Carmovirus; Caulimovirus; Carmovirus (Cavemovirus); the chlamydozoan minute phage; Chlorovirus; Chloriridovirus belongs to; chrysovirus; PCV-II belongs to; linear Tobamovirus; the coccolithus Tobamovirus; the colorado tick fever Tobamovirus belongs to (Coltivirus); Comovirus; coronavirus genus; corticovirus belongs to; cricket fiber crops illness Tobamovirus; the Flos Cucurbitae mosaic virus belongs to; Beet curly top virus belongs to; cypovirus; Cystovirus belongs to; cytomegalovirus belongs to; Cytorhabdovirus; Dianthovirus (Dainthovirus); the δ Papillomavirus; Deltaretrovirus; Densovirus; dependovirus; ebola virus belongs to; the prominent mosaic virus of Peas ear belongs to; enterovirus genus; insect biplate section RNA viruses belongs to; the A Entomopoxvirus; the B Entomopoxvirus; the C Entomopoxvirus; Ephemerovirus; the ε Papillomavirus; the ε retrovirus belongs to; equine rhinoviruses belongs to; equine rhinoviruses belongs to; Erythrovirus; the Δ Papillomavirus; broad bean wilt virus belongs to; Fijivirus belongs to; Flavivirus; the depression Tobamovirus; small fusiform phage; γ fat hair phage (Gammalipothrixvirus); the γ Papillomavirus; Gammaretrovirus; Giardiavirus; Granulovirus; little shape Tobamovirus (Guttavirus); Circovirus (Gyrovirus); Hantavirus; Hemivirus; prosperous Nipah virus belongs to; hepatitis virus belongs to; hepadnavirus belongs to; hepatovirus; Hypovirus; Ichnovirus Channel-catfish fish herpetovirus belongs to; reovirus belongs to; isometrical variable Tobamovirus; infectious laryngotracheitis virus belongs to; Influenza Virus A type; the Influenza Virus Type B; the influenza orthomyxovirus belongs to the C type; inovirus belongs to; ι Papillomavirus (Iotapapillomavirus); sweet potato viruses belongs to; iridescent virus; the infectious salmon anaemia Tobamovirus; repeat Tobamovirus; κ Papillomavirus (Kappapapillomavirus); the ridge Tobamovirus; Lagovirus; λ Papillomavirus (Lambdapapillomavirus); Leishmaniavirus; lentivirus; hare poxvirus belongs to, and levivirus belongs to; Luteovirus; lymph is lain concealed Tobamovirus; Lymphocystivirus; lyssavirus; corn chlorotic mottle poison belongs to (Machlomovirus); Macluravirus; the grape mottle virus belongs to; the Mammals Astrovirus; fork mandarin orange (oranges and tangerines) Tobamovirus (Mandarivirus); Marafivirus; Marburg virus belongs to; the Marek poison belongs to; Marnavirus; mastadenovirus; Mastrevirus; the enlargement cell virus belongs to; Metapneumovirus; Metavirus; microvirus belongs to; Mitovirus; Molluscipoxvirus; Morbillivirus; Mupapillomavirus; Muromegalovirus; fly reovirus genus (Mycoreovirus); the Na Yiluo Tobamovirus; nanometer Tobamovirus (Nanovirus); Narnavirus; Necrovirus; Nepovirus; the Nova Tobamovirus; the outer Rhabdovirus of grain; nucleopolyhedrosis virus belongs to; Nucleorhabdovirus; μ Papillomavirus (Nupapillomavirus); helmet Tobamovirus; bromovirus; Omegatetravirus; occasion type Papillomavirus; Orbivirus; positive Bunyavirus (Orthobunyavirus); positive liver DNA (desoxyribose nucleic acid) Tobamovirus; orthopoxvirus; Orthoreovirus; Oryzavirus (Oryzavirus); handkerchief Nico Tobamovirus (Panicovirus); parapoxvirus belongs to; the lonely Tobamovirus of secondary intestines; partitivirus belongs to (Patitivirus); parvovirus genus group; the Peroplaneta fluligginosa Densovirus; plague virus belongs to; morning glory leaf vein transparent sample Tobamovirus (Petuvirus); brown algal virus belongs to; Phlebovirus; Phytoreovirus; the π Papillomavirus; plasmavirus belongs to; Plectrovi; Pneumovirus; Polerovirus; Polyomavirus; potexvirus; Potyvirus; parasitic phycovirus belongs to; Prymnesiovirus; pseudovirus belongs to; Ranavirus; the needle phycovirus belongs to; Respirovirus; monkey Tobamovirus (Rhadinovirus); Rhinovirus; Roseolovirus; rotavirus; rubella virus genus; Rubulavirus; ancient phage; ryegrass mosaic virus belongs to; the bed ripples Tobamovirus; Seadornavirus; association Tobamovirus (Sequivirus); the sialidase Tobamovirus; Simplexvirus; mottle virus belongs to; the spirochete minute phage; Spumavirus; Suipoxvirus; tectivirus belongs to; prompt Shen Tobamovirus; θ Papillomavirus (Thetapapillomavirus); hold in the palm high native Tobamovirus; Tombusvirus; the pseudo-curly top poison of tomato belongs to; Orbivirus; tomato spotted wilf virus belongs to; totivirus belongs to; send out the shape Tobamovirus; the Triticum Tobamovirus; tungro bacilliform virus belongs to; Tymovirus; Varicellavirus; Vesiculovirus; the vesicular rass Tobamovirus; the grape Tobamovirus; stunt virus belongs to; white spot virus belongs to; ξ Papillomavirus (Xipapillomavirus); the virus of Yatapoxvirus or ζ Papillomavirus (Zetapapillomavirus).

[060] in another embodiment, the present invention relates to INS sequence in the genome of virus of Retroviridae.The example of this viroid includes but not limited to the virus of α retrovirus, β retrovirus, δ retrovirus, ε retrovirus, γ retrovirus, foamy virus and lentivirus.

[061] in other embodiments, the present invention relates to the INS sequence in the genome of slow virus.The example of this type of slow virus includes but not limited to bovine immunodeficiency virus, equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), goat sacroiliitis encephalitis, myelin come off-be short of breath virus, human immunodeficiency virus's 1 type (HIV-1), human immunodeficiency virus's 2 types (HIV-2) and simian immunodeficiency virus (SIV).

[062] the excessive appearance of AGG motif and its conservative property in lentiviridae are hinting that it is important on function.It is believed that the AGG sequence motifs can be the INS sequence, and can have retarding effect virus with it.For example, it is believed that AGG motif of the present invention can participate in usually owing to any of INS sequence and all retarding effects, include but not limited to keep viral RNA low steady state levels, slow down the turnover and possible the hiding of virus.

[063] discovery of AGG motif provides the evaluation of the production that is used for production of vaccine, recombinant viral proteins matter, new drug and has been used to study the new chance of hiding of virus or the like.Be described in more detail below this type of application.

[064] in one embodiment, thus having the present invention relates to carry out sudden change changes over the slow virus of non-AGG sequence or the nucleic acid of virus, for example HIV nucleic acid with one or more AGG sequences.In another embodiment, the present invention relates to produce the method for this type of sudden change.Can produce this type of sudden change in any position in the genome of slow virus or virus (comprising coding and non-coding region).For example, in one embodiment, sudden change can be present among gag, the pol and/or env gene of lentiviral gene group.Also can in any nucleic acid that derives from slow virus or virus, produce this type of sudden change.Thereby the present invention includes any and all derive from slow virus or virus, carried out sudden change with one or more AGG sequences change over non-AGG sequence nucleic acid and any and all produce the method for this type of sudden change, no matter whether this nucleic acid is present in virus, vaccine strain system, plasmid, expression vector or elsewhere with the free nucleic acid molecule.

[065] by AGG motif of the present invention can being mutated into any non-AGG sequence with the one or more Nucleotide in another nucleotide subsitution AGG motif.For example, can with first Nucleotide in the AGG motif from A be mutated into G, T, C, U or any other natural or synthetic Nucleotide.Can with second Nucleotide in the AGG motif from G be mutated into A, T, C, U or any other natural or synthetic Nucleotide.Can with the trinucleotide in the AGG motif from G be mutated into A, T, C, U or any other natural or synthetic Nucleotide.Can change any one site of AGG motif, maybe can change a plurality of sites of AGG motif.For example, can change nucleotide site 1,2 or 3 (wherein site 1 is to be originally that the site of A, site 2 were originally to be the site of G, and site 3 is originally to be the site of second G) in the AGG motif.In addition, any two sites of AGG motif can be changed as mentioned above, or all three sites of AGG motif can be changed as mentioned above.The AGG motif is mutated into the sudden change that non-AGG sequence also comprises other types, for example inserts one or more Nucleotide, or lack one or more Nucleotide and substitute other Nucleotide without them from AGG with destruction AGG motif.

[066] AGG motif to be changed can be positioned at any position of any slow virus, virus, the slow virus nucleic acid source or viral source, for example is positioned at coding or non-coding region.The AGG motif is arranged in the embodiment of coding region therein, the AGG motif can be changed over the amino acid whose sequence that does not change by described nucleic acid encoding.For example, constitute single codon, thereby under the situation of coded amino acid arginine (Arg), motif can be changed over AGA, CGG, CGA, CGC, CGU or CGT, its arginine of also encoding separately at the AGG motif.The AGG motif may be crossed over two codons in the coding region.If like this, may once more the AGG motif be changed over the sequence that needn't change any amino acids coding in two codons crossing over by the AGG motif.By reference genetic code or RNA or DNA password sublist, those skilled in the art can easily determine how to change the one or more nucleotide positions in the AGG motif and not change coded amino acid.

[067] in some embodiments, the AGG motif can be changed over and do not influence coded amino acid whose non-AGG trinucleotide.This type of sudden change can cause one or more different amino acid to be encoded, and maybe can cause one or more aminoacid deletion or be added into aminoacid sequence.If the AGG motif changed over does not influence coded amino acid whose non-AGG trinucleotide, may produce one that to influence in coded proteinic structure, function or the immunogenic a plurality of amino acid change sharply.For example, the mutain by the mutant nucleic acid coding can have the structure substantially the same with wild-type protein and/or function and/or immunogenicity.Some are amino acid whose to change and may be able to cause the immunogenicity that increases, those skilled in the art will know that when this type of modification is suitable.

[068] can produce of the sudden change of AGG motif by any suitable mutafacient system known in the art to non-AGG motif, described method includes but not limited to, the mutagenesis that site-directed mutagenesis, oligonucleotide instruct, positive microbiotic back-and-forth method, single restriction site null method (uniquerestriction site elimination, USE), the mutagenesis of deoxyuridine integration method (deoxyuridine incorporation), phosphorothioate integration method (phosphorothioateincorporation) and PCR-based.The detailed content of these class methods is found in for example people (1990) Nucl.Acids Res.18 such as Lewis, p3439; People such as Bohnsack (1996) Meth.Mol.Biol.57, p1; People such as Vavra (1996) Promega Notes58,30; Altered

II in vitro Mutagenesis Systems TechnicalManual#TM001, Promega Corporation; People such as Deng. (1992) Anal.Biochem.200, p81; People such as Kunkel (1985) Proc.Natl.Acad.Sci.USA82, p488; People such as Kunke (1987) Meth.Enzymol.154, p367; People such as Taylor (1985) Nucl.Acids Res.13, p8764; People such as Nakamaye (1986) Nucl.Acids Res.14, p9679; People such as Higuchi (1988) Nucl.Acids Res.16, p7351; People such as Shimada (1996) Meth.Mol.Biol.57, p157; People such as Ho (1989) Gene 77, p51; People such as Horton (1989) Gene 77, people such as p61 and Sarkar (1990) BioTechniques 8, p404.The many test kits that are used to carry out site-directed mutagenesis are commercially available acquisitions, for example available from Stratgene Inc.'s IISite-Directed Mutagenesis test kit and available from the Altered of Promega Inc

II vitro mutagenesis system.This type of is purchased obtainable test kit and also can be used for the AGG motif is mutated into non-AGG sequence.

[069] Vaccine

[070] method and composition of the present invention can be used for the production of vaccine especially.The a small amount of virion that in infectious cycle, produces, in conjunction with the ability that they keep in cell for a long time, limited slow virus for example HIV to immune exposure.This character is favourable for virus, but has influenced the ability that produces effective vaccine unfriendly.For example, be designed for host cells infected and the virus vaccines that duplicates therein can produce low-level offspring and keep " hiding " for a long time in host cell.Therefore, this type of vaccine can not trigger immune response and immunological memory effectively.As if similarly, the dna vaccination of encode one or more slow viruss or virus antigen is expressed low-level antigen in the host, thereby limited the effect of dna vaccination in producing immune response and immunological memory.

[071] discovery of AGG motif of the present invention has increased the possibility that produces mutated viruses, described mutated viruses has AGG motif still less, thus have the infectious cycle of protein expression, increase of encoding viral of steady state levels, increase of the viral RNA of increase and increase to immune exposure.This type of mutated viruses can be used as virus vaccines.Be described in more detail below the vaccine that comprises or derive from this type of mutated viruses.The discovery of AGG motif of the present invention has also increased the possibility that produces the mutated viruses nucleotide sequence, and described nucleotide sequence is with than much higher in other cases speed (rate) and/or the much bigger sick malicious encoded protein matter of volume production.This type of mutant nucleic acid can be used as dna vaccination, as is described in greater detail below.In addition, this type of mutant nucleic acid also can be used for producing the virus protein that is used for protein vaccine.Also described in more detail below and comprised or the vaccine that produces of proteinoid from then on.

[072] the present invention includes and prevent/preventing property vaccine and therapeutic vaccine.Preventative vaccine is the vaccine that the experimenter who does not catch is used, and described vaccine designs so that protection to be provided at described disease.The ideal preventative vaccine will stop virus to cause infection in the experimenter of inoculation, and promptly it will provide protective immunity completely.Yet even it does not provide protective immunity completely, preventative vaccine still can give the experimenter some provide protections.For example preventative vaccine can reduce symptom, seriousness and/or the time length of disease.Under the situation of HIV, even be not enough to provide protective immunity completely at it, preventative vaccine can stop or postpone the progress of typical acquired immune deficiency syndrome (AIDS) (full blown AIDS).The administering therapeutic vaccine is to reduce the influence of virus infection in infecting this viral experimenter.Therapeutic vaccine can reduce symptom, seriousness and/or the time length of disease.Under the situation of HIV, therapeutic vaccine use the progress that can stop or postpone typical acquired immune deficiency syndrome (AIDS).

[073] the present invention includes any and all types of vaccine, described vaccine comprises the nucleic acid of the AGG motif with sudden change or produces from the nucleic acid of AGG motif with sudden change.Herein disclosed is several our dissimilar vaccines.Yet, one skilled in the art will realize that spendable vaccine that has other types and the additive method that is used to produce vaccine.The present invention is not limited to the vaccine of illustrational particular type.On the contrary, it comprises that any and all vaccines, described vaccine comprise the nucleic acid of the AGG motif with sudden change or produce from the nucleic acid of AGG motif with sudden change.

[074] the present invention includes " virus vaccines ".As used herein, term " virus vaccines " comprises the virus vaccines and the vector-viral vaccine of attenuated virus vaccine, deactivation.The present invention also comprises dna vaccination and protein properties or " subunit " vaccine, and it is described separately hereinafter.Should be pointed out that existence is significantly overlapping between dissimilar vaccines.For example, virus vaccines can comprise and the same or analogous nucleic acid of nucleic acid that is used to prepare dna vaccination.Similarly, dna vaccination and virus vaccines can be expressed and the same or analogous protein of protein that is used to prepare proteinaceous vaccine.Therefore, the description that is provided of the vaccine of any type should not be interpreted as only being used for this vaccine type.On the contrary, can be used for and be used to interchangeably any and all types of vaccine that the present invention includes all about the description of the vaccine of any type.

[075] in some aspects, the invention provides the immunogenic composition that to induce the anti-immune response that comprises the virus of slow virus of the present invention, comprise SEQ ID NO:1.In one embodiment, described immunogenic composition can improve the symptom of slow virus or virus infection and/or the time length of minimizing slow virus or virus infection.In another embodiment, described immunogenic composition can the malicious protective immunity that infects of inducing anti-disease.Immunogenic composition of the present invention can resist slow virus disclosed herein effectively, can be cross reaction and anti-effectively described virus and anti-other viruses with a plurality of different clades and the strain of slow virus also.

[076] Virus vaccines

[077] A) attenuated virus vaccine

[078] in one embodiment, the invention provides the attenuated virus vaccine of AGG sequence with one or more sudden changes.Attenuated virus is such virus, thus promptly described virus carried out changing they weakened, they just no longer cause disease like this, but but still immune response stimulating.Existence can make many methods of viral attenuation.For example, can coding be kept perfectly by the antigenic sequence of immune system recognition make viral attenuation by removing or destroy the virus sequence that causes that disease is required.Attenuated virus can or can not duplicate in host cell.The attenuated virus that can duplicate is useful, because virus amplification in vivo after the experimenter is used, thereby increased the immunogenic amount that can obtain to be used for immune response stimulating.

[079] according to the present invention, can obtain or produce suitable attenuated viral strains, the one or more AGG series jumps in the described attenuated viral strains become non-AGG sequence.Shown that several attenuated live virus vacciness are used to be protected from slow virus or virus infection.For example, the attenuation simian immunodeficiency virus (SIV) of living has been used to protect primates to avoid the attack of SIV.Referring to, for example, people such as Daniel, " Protective effects of a liveattenuated SIV vaccine witha deletion in the nef gene " (1992) Science 258, p1938; People such as Almond, " Protection by attenuated simian immunodeficiency virusin macaques against challenge with virus-infected cells " (1995) Lancet 345, p1342.Disclosed method of attenuating and attenuated viral strains can be used in combination with the present invention in these bibliographys.The additive method of attenuation is by people such as Desrosiers (" Identification of highly attenuated mutants of simianimmunodeficiency virus " (1998) J.Virol.72, p1431) and people such as Guan (" Construction and in vitro properties of a series ofattenuated simian immunodeficiency viruses with all accessorygenes deleted " (2001) J.Virol.75 p4056) describes.Should be pointed out that SIV is the common model of HIV, the method for attenuating that is used for SIV also can be used for HIV.The patent application WO/2001/007637 that announces has described modified attenuation HIV vaccine alive that duplicates under the situation that tetracycline analogue exists.Multiple other attenuation HIV alive strains have been developed, for example for " δ 4 " of the HIV-1 that lacks nef, vpr, vpu and Nef-response element or NRE gene with based on δ 4 vaccine strains but " the δ kURN " that in the gene of coding NFkB binding member, have extra disappearance.Also have several pieces of papers of describing the method that produces the attenuation HIV vaccine of living.Referring to for example, people such as Mills " Live attenuated HIV vaccines:a proposal forfurther research and development. " (2000) AIDS Res HumRetroviruses 16, p1453.Any these class methods that can be used for attenuation according to the present invention.If the method for attenuating that uses relates in the viral genome or the disappearance in the viral nucleic acid, can produce these sudden changes so, make it even as big as reducing accidental answer (chance reversion).If for example use these class methods, can lack 20 bases or polybase base more.

[080] B) killed virus vaccines

[081] in another embodiment, the invention provides " killing " or " deactivation " virus vaccines of the AGG sequence with one or more sudden changes.This type of vaccine is normally non-functional, thus do not express virogene or the inoculation the experimenter in do not duplicate.Yet method of the present invention is used in inactivation of viruses before in external or stripped amplification and the growth that promotes virus.For example, when the one or more AGG motifs in the virus being mutated into non-AGG sequence, the amplification rate of virus can increase, thereby produces more substantial virus, then with its deactivation with as vaccine.

[082] any suitable ablation method known in the art can be used for deactivation mutated viruses of the present invention, for example chemistry, heat or physical deactivation method or the deactivation undertaken by the radiation of using ionizing rays.For example, people such as Ilyinskii have developed the physical deactivation method of HIV, described method does not comprise its tertiary structure and possible immunogenic side's examination so that it becomes non-infectious, and using gas breaks/and the break virus structure is (referring to people such as Ilyinskii " Development of an Inactivated HIV Vaccine " (2001) AIDS VaccineSep 5-8; Abstract no.192).Other people have developed by using 0.2% beta-propiolactone (BPL) chemical ablation HIV virus to keep its immunogenic method (referring to people such as Addawe " Chemicallyi nactivated whole HIV vaccine induces cellularresponses in mice " (1996) Int Conf AIDS Jul 7-12 simultaneously; 11:4; Abstractno.Mo.A.100).The HIV vaccine of complete-deactivation is also tested in people's test.For example, in the patient that HIV infects, will make up the therapeutic vaccine that carries out deactivation by chemical treatment and radiating

(be also referred to as " HIV-1 immunogen ", " Salk Vaccine " or " AG1661 ") as immunotherapy carried out studying (referring to, people such as Fernandez-Cruz " 5-year evaluation of a therapeutic vaccine (HIV-1immunogen) administered with antiretrovirals in patients with HIV chronicinfection:induction of long-lasting HIV-specific cellularimmunity that impact on viral load " (2003) Second InternationalAIDS Society Conference on HIV Pathogenesis and Treatment, Paris, abstract 486).Method of the present invention can be used in combination with any above-mentioned ablation method or other virus inactivating methods known in the art.

[083] C) vector-viral vaccine

[084] thus also can one or more AGG series jumps be become the nucleotide sequence of the slow virus of the present invention of non-AGG sequence or virus to be integrated into to be suitable for the virus vector that the experimenter is used with having carried out sudden change.Slow virus or virus any slow virus of nucleic acid codified or virus protein, include but not limited to GAG, p17MA, p24CA, p7 and p6, GAG-POL, RT, RNAase H, PR, IN, Gp160, Gp120ENV, Gp41, Tat, Rev, Vpu, Vif, Vpr and Nef with and fragment, variant, homologue and derivative.The example of suitable virus vector includes but not limited to vaccinia virus (for example modified vaccinia virus Ankara or " MVA " are used for the height minus strain of the cowpox of antismallpox vaccine), retrovirus, Poxviruses (comprising canary pox, cowpox and fowl pox) adenovirus and adeno associated virus.Compare with their natural viral homologue, can change these virus vector, for example can carry out attenuation and/or they are become nonreplicative them.

[085] those skilled in the art can easily select the virus vector that is fit to and mutant nucleic acid of the present invention is inserted in such carrier.Described mutant nucleic acid should be under the control of the suitable promotor that the experimenter who is used for instructing slow virus or viral protein inoculating expresses.Can use the promotor that has been present in the virus vector.Alternatively, can use exogenous promoter.The example of suitable promotor includes but not limited to cytomegalovirus (CMV) promotor, Rous sarcoma virus (RSV) promotor, HIV long terminal repetition (HIV-LTR), HTLV-1LTR (HTLV-LTR) and hsv (HSV) thymidine kinase promoter.

[086] can be used for the technology that nucleotide sequence of the present invention inserts in the virus expression carrier is known to those skilled in the art.Referring to people (2001) Molecular Cloning:A Laboratory Manual such as for example Sambrook, the 3rd edition, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y (" Sambrook ").

[087] method of the present invention also can be used in combination with the vector-viral vaccine of any kind known in the art or with the improvement of the described vector-viral vaccine of opposing.The example of HIV vector-viral vaccine at present just under development comprise the Merck that comprises HIV clade B GAG-POL Nef non-replicability adenovirus carrier, comprise clade B Env, GAG, Pro, RT and Nef Sanofi Pasteur the canary pox carrier and comprise the MVA carrier of the Therion of clade B Env and GAG.At present the details of these and other the HIV vaccines in just developing by Www.hvtn.orgHIV Vaccine Trials Network provide.Method of the present invention can be used for improving the effect of vector-viral vaccine (for example be mutated into non-AGG sequence by the inhibition AGG motif with the nucleic acid component of slow virus or virus, thereby cause improving the vector-viral vaccine of the expression of protein in the experimenter of inoculation of slow virus or virus).

[088] Dna vaccination

[089] the present invention also comprises and is suitable for dna vaccination that the experimenter is used.The nucleic acid (the one or more AGG sequences with described nucleic acid change over non-AGG sequence) of the protein of any slow virus of coding or virus or the slow virus of its part, fragment, derivative or homologue or virus can be inserted DNA plasmid or expression vector to produce according to dna vaccination of the present invention.For example, in one embodiment, described dna vaccination comprise contain one or more coded proteins, one or more AGG sequences are changed over the plasmid of nucleic acid of the adenovirus or the virus of non-AGG sequence, described protein be selected from GAG, p17MA, p24CA, p7 and p6, GAG-POL, RT, RNAase H, PR, IN, Gp160, Gp120ENV, Gp41, Tat, Rev, Vpu, Vif, Vpr and Nef, with and fragment, variant, homologue and derivative.

[090] those skilled in the art can easily select suitable DNA plasmid or expression vector and mutant nucleic acid of the present invention is inserted in the such plasmid or expression vector.The proteinic nucleic acid of coding slow virus or virus should be under the control of the suitable promotor that the experimenter who is used for instructing the nucleic acid that one or more AGG sequences changed over non-AGG sequence in inoculation expresses.Can use the promotor that has been present in the expression vector.Alternatively, can use exogenous promoter.The example of suitable promotor includes but not limited to cytomegalovirus (CMV) promotor, Rous sarcoma virus (RSV) promotor, HIV long terminal repetition (HIV-LTR), HTLV-1LTR (HTLV-LTR) and hsv (HSV) thymidine kinase promoter.

[091] can be used for the technology that nucleotide sequence of the present invention inserts in DNA plasmid and the expression vector is known to those skilled in the art.For example, spendable standard recombinant dna technical description is in Sambrook.

[092] method of the present invention also can with the slow virus of any kind known in the art or virus DNA vaccine be used in combination or with the improvement of the described virus DNA vaccine of opposing.The DNA plasmid of the Chiron that the example of dna vaccination at present just under development comprises the NIH DNA plasmid that comprises clade B Gag, Pol, Nef and clade A, B and C, Env, comprise clade B Gag and Env and be the GENEVAX of the DNA plasmid that comprises clade B Gag.The details of these and other HIV vaccines at present just under development by Www.hvtn.orgHIV Vaccine Trials Network (HVTN) provide.Method of the present invention can be used for improving the effect of vaccine (for example be mutated into non-AGG sequence by the inhibition AGG motif with the nucleic acid component of slow virus or virus, thereby cause improving the vaccine of the expression of protein in the experimenter of inoculation of slow virus or virus).

[093] Protein vaccine

[094] the present invention also comprises protein vaccine.Protein south, its fragment, derivative, variant or the homologue of any slow virus or virus can be used for producing according to protein vaccine of the present invention.For example, in one embodiment, select to be selected from nucleic acid and its fragment, variant, homologue and the derivative of coding GAG, p17MA, p24CA, p7 and p6, GAG-POL, RT, RNA enzyme H, PR, IN, Gp160, Gp120ENV, Gp41, Tat, Rev, Vpu, Vif, Vpr and Nef with the nucleic acid of the slow virus of modifying or virus by method of the present invention.The favourable aspect of using method of the present invention to produce this type of vaccine is by one or more AGG motifs being mutated into non-AGG sequence, can significantly increasing the proteinic amount of generation and/or the speed that protein produces.

[095] in one embodiment, the nucleic acid of the slow virus that will modify by method of the present invention or virus is integrated into suitable expression vector and expresses in appropriate expression system to allow described protein.The example of appropriate expression system includes but not limited to mammalian cell, insect cell, bacterial cell or the yeast cell cultivated.Can in cell, express the protein of slow virus or virus or peptide then and it is carried out purifying.The protein of purifying can be integrated into then and be suitable for composition that the experimenter is used.The method and the technology that are used to express with purification of recombinant proteins matter are known in this area, can use any this type of suitable method.

[096] can use any plasmid or expression vector, as long as it comprises the protein expression promoter in the expression system of hope that instructs slow virus or virus.For example, if in bacterial cell, produce protein, should use so and can in bacterium, instruct expression promoter, if in mammalian cell, produce protein, should use so and can in mammalian cell, instruct expression promoter,, should use so and can in insect cell, instruct expression promoter if in insect cell, produce protein, if in yeast, produce protein, should use so and can in yeast, instruct expression promoter.In another embodiment, in mammalian expression system from the mammalian promoter marking protein.Suitable promotor includes but not limited to cytomegalovirus (CMV) promotor, Rous sarcoma virus (RSV) promotor, HIV long terminal repetition (HIV-LTR), HTLV-1LTR (HTLV-LTR), hsv (HSV) thymidine kinase promoter and SV40 virus early promoter.Suitable expression vector includes but not limited to that clay, plasmid and virus vector be replication defect type retrovirus, adenovirus, adeno associated virus, simplexvirus, vaccinia virus, attenuated vaccinia virus, canary pox virus, slow virus and simplexvirus or the like for example.Also can use the expression vector that has comprised the suitable promotor and the commercially available acquisition of the cloning site that is used for the exogenous nucleic acid adding.

[097] can use any appropriate expression system, for example bacterium, yeast, insect or mammalian cell expression system.In another embodiment, expression slow virus or viral protein in the nucleic acid stability ground of the slow virus of sudden change of the present invention or virus or instantaneous ground mammalian cells transfected.The example of spendable suitable mammalian cell includes but not limited to COS, CHO, BHK, HEK293, VERO, HeLa, MDCK, WI38 and NIH 3T3 cell.Directly from Mammals obtain through transforming with former generation of comprising mutant nucleic acid of the present invention or inferiorly also can be used as expression system for cell.

[098] those skilled in the art can easily select appropriate expression system, promotor and expression vector used according to the present invention.The case description of the combination of spendable clone and expression vector is in Sambrook.Can be used for the technology of nucleotide sequence insertion expression vector of the present invention is known to those skilled in the art.Referring to, for example, Sambrook.

[099] method of the present invention also can be used in combination with the protein vaccine of any kind known in the art or with the improvement of the described protein vaccine of opposing.The example of protein vaccine at present just under development comprises the protein subunit clade B Env that comprises Chiron and clade B Nef-Tat fusion rotein and the clade B Env subunit of GlaxoSmithKline).Method of the present invention can be used to improve the production effect of vaccine (for example being mutated into the vaccine of non-AGG sequence by the inhibition AGG motif in the nucleic acid of the range protein subunit of will encoding).

[0100] immunogenic composition of the present invention can comprise subunit vaccine.Subunit vaccine comprises for example dna vaccination of nucleic acid vaccine, and described nucleic acid vaccine comprises one or more virus proteins of coding or subunit, the perhaps nucleic acid of the part of these protein or subunit.When using this type of vaccine, use described nucleic acid to the experimenter, in the experimenter, express immunogenic protein or peptide, thereby in described experimenter, produced the immune response of anti-described protein or peptide by nucleic acid encoding.Subunit seedling also can be a protein vaccine, itself comprises the protein or the subunit of virus, perhaps the part of this proteinoid or subunit.Subunit vaccine codified of the present invention or comprise any slow virus disclosed herein or virus protein or peptide or in the experimenter, have immunogenic its any part, fragment, derivative or mutant.Those skilled in the art can easily test slow virus described herein or the protein of virus and the immunogenicity of peptide, and can select suitable protein or peptide to be used for subunit vaccine.

[0101] Vaccine composition

[0102] vaccine composition of the present invention comprises at least a virus (virus and the virus vector that comprise attenuated virus, deactivation), nucleic acid or protein, and is for example mentioned above.Described composition also can comprise one or more extra components, includes but not limited to pharmaceutically acceptable carrier, buffer reagent, stablizer, thinner (for example water), sanitas, solubilizing agent or immunomodulator.Suitable immunomodulator includes but not limited to promote at least one component of vaccine of the present invention by the polynucleotide and the reagent of the adjuvant of immune system recognition, cytokine, the Codocyte factor.Those skilled in the art can easily select suitable additive to be included in the vaccine composition of the present invention.

[0103] carrier of hydrophobic compound of the present invention can be comprise phenylcarbinol, non-polar surfactant, can with the miscible organic polymer of water and the cosolvent system of water.Described cosolvent system can be a VPD cosolvent system.VPD is that non-polar surfactant's polysorbate80 and the 65%w/v Liquid Macrogol of 3%w/v phenylcarbinol, 8%w/v mixes the solution of forming in dehydrated alcohol.VPD cosolvent system (VPD:5W) is by forming with 5% the D/W VPD with dilution in 1: 1.The abundant solubilizing hydrophobic compound of this cosolvent system, and itself produces hypotoxicity after general is used.Natively, sizable change can take place and not destroy its solvability and toxic characteristic in the ratio of cosolvent system.In addition, itself can change the cosolvent component: for example, can use other hypotoxicities non-polar surfactant but not polysorbate80; The fraction size of polyoxyethylene glycol can change; The other biological compatible polymer can replace polyoxyethylene glycol, for example polyvinylpyrrolidone; And other sugar or the alternative glucose of polysaccharide.Alternatively, can use other delivery systems that are used for hydrophobic immunogenic compound.Liposome and emulsion are the examples of knowing that is used for the delivery vehicle of dewatering medicament or carrier.Also can use for example methyl-sulphoxide of some organic solvent, although usually with bigger toxicity cost.In addition, the semipermeability matrix that can use sustained release system for example to comprise the solid hydrophobic polymkeric substance of therapeutical agent is sent compound.Set up polytype lasting releasable material, described material is known to those skilled in the art.According to their chemical property, lasting release capsule can continue to discharge compound from many days several weeks to 100.According to the chemical property and the biological stability of therapeutical agent, can use the other strategy that is used for stabilizing protein or other activeconstituentss.

[0104] described immunogenic composition also can comprise suitable solid phase or gel phase carrier or vehicle.The example of examples of such carriers or vehicle includes but not limited to for example polyoxyethylene glycol of lime carbonate, calcium phosphate, various sugar, starch, derivatived cellulose, gelatin and polymkeric substance.Can provide many activeconstituentss of the present invention with the form of the salt of the counterion compatible with immunogenicity.The acceptable base addition salt of this type of immunity be keep the biological effectiveness of free acid and character salt and by with the inorganic or organic bases salt that obtains of reactions such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, an alkylamine, dibasic amino acid (dibasic amino acid), sodium-acetate, potassium benzoate, trolamine for example.

[0105] immunogenic composition of the present invention can be protein of the present invention or other activeconstituentss composite form with protein or peptide antigen.Protein and/or peptide antigen are delivered to bone-marrow-derived lymphocyte and T lymphocyte with stimulus signal.Bone-marrow-derived lymphocyte will be by their expression immunoglobulin receptor response antigen.The T lymphocyte will be after the antigen presentation of being undertaken by MHC albumen, by TXi Baoshouti (TCR) response antigen.MHC and structurally associated protein (comprising on the host cell by I class and II class mhc gene encoded protein matter) are used for the peptide antigen presentation to the T lymphocyte.Can also be with independent or provide antigen component with the MHC peptide complex form of the purifying of the costimulatory molecules that can be directly signals to the T cell.Alternatively, can will be able to and can mix with immunogenic composition of the present invention in conjunction with the antibody of the surface immumoglobulin on the B cell and other molecules in conjunction with the antibody of the TCR on the T cell and other molecules.

[0106] form that immunogenic composition of the present invention can liposome exists, in described liposome, protein of the present invention with, except that other acceptable carriers, the amphipathic reagent that exists with accumulative form such as micelle, insoluble monolayer, liquid crystal or laminated layer form in the aqueous solution for example lipid mixes.The suitable lipid that is used for Liposomal formulation includes but not limited to monoglyceride, triglyceride, sulfatide, lysolecithin, phosphatide, saponin(e, bile acide etc.The preparation of this type of Liposomal formulation is within those skilled in the art's level, as at for example U.S. Patent number 4,235,871; 4,501,728; 4,837,028 and 4,737,323 is disclosed, and it all integrates with this paper by reference.

[0107] other additives that are used for vaccine preparation are known and it will be apparent to those skilled in the art that.

[0108] Significant quantity

[0109] should the experimenter be used the vaccine composition of the present invention of " immune significant quantity ".As used herein, term " immune significant quantity " is meant and can induces the immune response of hope or strengthen described immunoreactive inductive amount in the experimenter.The reaction of described hope can comprise, among other things, induces antibody or cell-mediated immune response or both, reduces virus load, improves symptom, the generation that postpones symptom infected, reduces time length of infecting etc.Described immune significant quantity can also be the amount of effectively inducing protective immunity.

[0110] those skilled in the art can easily determine what is " immune significant quantity " and need not the over-drastic experiment.For example, can determine significant quantity by ordinary method (from low dosage, constantly increase dosage then, monitor immunological effect simultaneously).When the optimum quantity determining to use, can consider many factors, the existence of stature size, age and general physical condition, other vaccines or the medicine that comprises the experimenter in the experimenter, inoculate with the toxicity of the specific virus of opposing to the experimenter etc.Can behind the result who considers from the different experiments zooscopy, select actual dose.

[0111] Send/approach of application program

[0112] can use vaccine composition of the present invention with single agent, multi-agent or use " initial-as to strengthen " scheme.Initial when using-during strengthened scheme, vaccine of the present invention can be used as " initially " agent or " reinforcement " agent or both.Can be by the combined administration composition of any suitable way or approach, described approach includes but not limited to parenteral, intracutaneous, in skin, subcutaneous, intramuscular, intravenously, intraperitoneal, nose, oral or intraocular approach.Also can use with wrapped on it by the particle of composition of the present invention for example gold grain inject the experimenter skin " rifle " device applying said compositions.Those skilled in the art can prepare described vaccine composition according to the route of delivery of selecting.

[0113] Viral purification

[0114] method of the virus of purifying deactivation is known in the field and can comprises for example one or more methods in ion exchange chromatography, size exclusion chromatography and the liquid affinity chromatography of for example gradient centrifugation, ultracentrifugation, continuous flow ultracentrifugation (continuous-flowultracentrifugation) and chromatography.Other purification process comprises ultracentrifugation and dialfiltration.Referring to JP Gregersen " Herstellung vonVirussimpfstoffen aus Zellkulturen " Chapter 4.2inPharmazeutische Biotecnology (eds.O.Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.Also referring to, people such as O ' Neil, " Virus Harvesting and Affinity Based LiquidChromatography.A Method for Virus Concentration andPurification ", Biotechnology (1993) 11:173-177; People such as Prior, " Process Devel opment for Manufacture of Inactivated HIV-1 ", Pharmaceutical Technology (1995) 30-52; With people such as Ma jhdi, " Isolation and Characterization of a Coronavirus from ElkCalves with diarrhea " Journal of Clinical Microbiology (1995) 35 (11): 2937-2942.

[0115] other examples that are suitable for purification process of the present invention comprise that polyoxyethylene glycol or ammonium sulfate precipitation are (referring to people such as Trepanier, " Concentration of humanrespiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration " Journalof Virological Methods (1981) 3 (4): 201-211; People such as Hagen, " Optimization of Poly (ethylene glycol) Precipitation ofHepatitis Virus Used to prepare VAQTA, a Highly PurifiedInactivated Vaccine " Biotechnology Progress (1996) 12:406-412; With people such as Carlsson, " Purification of Infectious PancreaticNecrosis Virus by Anion Exchange Chromatography Increases theSpecific Infectivity " Journal of Virological Methods (1994) 47:27-36) and ultracentrifugation and microfiltration (referring to people such as Pay, Developments in Biological Standardization (1985) 60:171-174; People such as Tsurumi, " Structure and filtration performances ofimproved cuprammonium regenerated cellulose hollow fibre (improved BMM hollow fibre) for virusr emoval " Polymer Journal (1990) 22 (12): 1085-1100; With people such as Makino, " Concentration of liveretrovirus with a regenerated cellulose hollow fibre, BMM ", Archives of Virology (1994) 139 (1-2): 87-96.).

[0116] can use for example ion exchange chromatography purified virus of chromatography.Chromatography purification allows to produce the suspension that comprises virus of large volume.Can purpose viral product and chromatography media be interacted by simple absorption/go absorption mechanism, can in single load, handle bulk sample then.The pollutent that does not have avidity for sorbent material passes through pillar.Then with spissated form wash-out viral material.

[0117] spendable anionite-exchange resin comprises DEAE, EMD TMAE.Spendable Zeo-karb can comprise the surface that sulfonic acid is modified.Can use and comprise reinforcing yin essence ion exchange resin (for example EMD TMAE) that is used for first step and the ion exchange chromatography purified virus that is used for the EMD-SO.sub.3 (Zeo-karb) of second step.Melts combine affinity chromatography step can randomly be used to be further purified.(referring to, for example, WO97/06243).

[0118] also can use for example Fractogel.TM.EMD. of resin.The long linear polymer chain (so-called " antenna " (tentacle)) that should have covalent attachment based on the resin of synthetic methacrylic acid ester.Should " antenna chemical substance " allow accessible part binding biomolecules on a large amount of spaces and not have any sterically hindered.This resin also has the pressure stability of raising.

[0119] the liquid affinity chromatography based on pillar is to can be used for another kind of purification process of the present invention.An example that is used for the resin of purification process is Matrex.TM.Cellufine.TM.Sulfate (MCS).MCS is made up of hard spherical (the approximately diameter of 45-105 μ m) cellulose matrix (its pore structure exclusion macromole) of 3,000 daltonian exclusion limits, and described matrix has the sulfuric ester functional group of lower concentration on cellulosic 6 positions).When sense part (sulfuric ester) relative height spread, it presented insufficient cationic charge density, thereby most of soluble protein is adsorbed on the surface of beads.Therefore, wash out a large amount of protein (cell culture supernatant liquid, for example pyrogen and most of contaminating protein matter, and nucleic acid and intracellular toxin) of typical virus base, finding, thereby obtain bonded virus purifying to a certain degree from pillar.

[0120] the hard high-intensity bead of MCS tends to incompressible.Pressure/the flow performance of MCS resin allows high linear rate of flow, and then allows even carry out high speed processing in large pillar, makes it become (scalable) unit operation that is easy to upgrade.In addition, use the chromatography purification step of MCS to provide increase to guarantee safety and product sterility, thereby avoid operation of over-drastic product and security consideration.When intracellular toxin does not combine with it, the MCS purification step allow fast and free of contamination reduce phlegm and internal heat former.Gentle combination and elution requirement provide heavy body and product output.Therefore the MCS resin represent to be used to concentrate, purifying and reduce phlegm and internal heat former simple, fast, effectively and the method for saving cost.In addition, can repeatedly use the MCS resin once more.

[0121] virus of deactivation can be further purified by gradient centrifugation or density gradient centrifugation.About the commercial size operation, the sucrose gradient centrifugation of continuous flow can be a selection.This method is widely used in the purifying antiviral vaccine and is known (referring to J P Gregersen " Herstellung von Virussimpfstoffen ausZellkulturen " Chapter 4.2 in Pharmazeutische Biotechnology (eds.O.Kayser and R H Mueller) WissenschaftlicheVerlagsgesellschaft to those skilled in the art, Stuttgart, 2000.).

[0122] other purification process that can be used for purifying virus of the present invention comprise nucleic acid-degrading agent, the nucleolysis enzyme (nuclease that for example has DNA enzyme and RNA enzymic activity, perhaps endonuclease, for example from serratia marcescens (Serratia marcescen) as the commercially available endonuclease of Benzonase.TM.), have anionic functional group's film adsorber (for example Sartobind.TM.) or use the use of anionic functional group's (for example DEAE or TMAE) extra chromatographic step.Also can in purification process, add ultrafiltration/dialfiltration and final sterile filtration step.

[0123] the viral prepared product of purifying of the present invention is substantially devoid of the contaminative protein that derives from cell or cell culture, it can comprise and is lower than about 1000,500,250,150,100 or 50pg nucleus/μ g virus antigen and be lower than about 1000,500,250,150,100 or 50pg nucleus/dosage.The viral prepared product of purifying also can comprise and is lower than about 20pg or is lower than about 10pg.The method of measuring host cell nucleic acid level in the viral sample is known in this area.Can use for example WHO or the standardized method FDA approval or that recommend by supervisory and management department.

[0124] Other embodiments of the present invention

[0125] in other embodiments, the present invention relates to be used to identify the reagent of the generation of the generation of the generation that suppresses or stimulate viral RNA, virus protein or virion, perhaps suppress or stimulate the compositions and methods of hiding of virus.In another embodiment, described method comprises to be provided the control cells that comprises at least one nucleic acid sequence that contains at least one AGG motif and comprises the subject cell that at least one contains the nucleic acid sequence of at least one AGG motif that has been mutated into non-AGG sequence, subject cell is contacted with one or more reagent with control cells, and identify at least a, compare with control cells, in subject cell, suppress or stimulate the generation of viral RNA, the reagent of the generation of virus protein or the generation of virion, the perhaps reagent of hiding of inhibition or stimulation virus.

[0126] in other embodiments, the present invention relates to be used to identify the reagent that suppresses or stimulate proteinic generation of generation, slow virus or the generation of slow virus particulate of slow virus RNA, the compositions and methods of hiding that perhaps suppresses or stimulate slow virus.In another embodiment, described method comprises the control cells that the nucleotide sequence that comprises at least one slow virus that contains at least one AGG motif or virus is provided and comprises at least one slow virus that contains at least one AGG motif that has been mutated into non-AGG sequence or the subject cell of the nucleotide sequence of virus, subject cell is contacted with one or more reagent with control cells, and identify at least a, compare with control cells, in subject cell, suppress or stimulate the generation of slow virus or viral RNA, the reagent that the particulate of the proteinic generation of slow virus or virus or slow virus or virus produces, the perhaps reagent of hiding of inhibition or stimulation slow virus or virus.

[0127] in some embodiments, described reagent suppresses or stimulates the proteinic generation of generation, HIV or generation of HIV particulate or the inhibition of HIV RNA or stimulate HIV to hide.For example, can use whole " library " of high-throughput screening method with this mode screening reagent.Those skilled in the art can easily design the reagent that high-throughput screening method produces with the particulate of the proteinic generation of the generation, slow virus or the virus that identify to suppress or stimulate slow virus or viral RNA or slow virus or virus, the reagent of hiding that perhaps suppresses or stimulate slow virus.Be used for knowing in the method for porous plate culturing cell, the different compositions and methods that are used for using from the reagent library to the different holes of porous plate are known.Several method can be used for determining the influence that library reagent produces the particulate of the proteinic generation of generation, slow virus or the virus of slow virus or viral RNA or slow virus or virus, perhaps to the influence of hiding of slow virus or virus.For example, the cell that is used for high flux screening can be transformed with one or more fusion roteins of encoding, for example the protein and for example fusion between the green fluorescent protein (GFP) of fluorescin of slow virus or virus.Like this, can monitor the proteinic generation of slow virus or virus by fluorescence detection method, the reagent of the proteinic generation of the feasible stimulation of described detection method or inhibition slow virus or virus can be detected.

[0128] in another embodiment, the present invention relates to be used to identify the method for AGG motif wedding agent.In one embodiment, described method comprises the nucleic acid that tried that the contrast nucleic acid that comprises at least one AGG motif is provided and comprises at least one AGG motif that has been mutated into non-AGG sequence, to be tried nucleic acid contacts with one or more reagent with contrast nucleic acid and identifies and at least aly tried the reagent of nucleic acid or to combine the reagent that contrasts nucleic acid than its avidity higher with being tried nucleic acid bonded avidity in conjunction with contrast nucleic acid but debond.In another embodiment, described method comprises provides the contrast nucleic acid that is tried nucleic acid and combination at random that comprises Nucleotide and order that comprises a plurality of repetition AGG motifs, to be tried nucleic acid contacts with one or more reagent with contrast nucleic acid and identifies that at least a combination is tried nucleic acid but debond is tried to contrast the reagent of nucleic acid or with than it and contrast the higher avidity of nucleic acid bonded avidity and combine the reagent that is tried nucleic acid.Existence can detect the many methods in conjunction with the reagent of these structures.For example, in one embodiment, can provide the above-mentioned nucleic acid that tried to pass through these matrix on the pillar or on some other suitable solid substrates with contrast nucleic acid with given the test agent (for example library of the cell lysate or the agent of being had a try).But wash-out and analysis are in conjunction with the reagent that is tried material and/or contrast material.In other embodiments, the yeast one-hybrid method can be used for identifying the reagent in conjunction with the AGG motif.In other embodiments, can carry out electrophoretic mobility shift assay (EMSA) to identify reagent in conjunction with the AGG motif.Be suitable for identifying that the additive method of Nucleotide wedding agent is known in this area, and any these class methods can be used for identifying the reagent in conjunction with the AGG motif.The present invention also comprises AGG motif wedding agent, for example uses the AGG motif wedding agent of the evaluation of method of the present invention.

[0129] in another embodiment, the present invention relates to suppress or stimulate the bonded reagent of AGG wedding agent and the nucleic acid that comprises at least one AGG motif and be used to identify this type of compositions and methods as mentioned above.

[0130] these and other embodiments of the present invention are further described among the following non-sex-limited embodiment.

Embodiment

[0131] Embodiment 1-identifies the AGG motif in the HIV-1 genome

[0132] because genetic code is a degeneracy, thus nucleotide sequence can be on nucleotide level different mutually but encode identical protein or peptide.Yet,, have selection pressure usually for selection of specific cryptosystem and AT/GC content at occurring in nature.Also exist selection pressure by amino acid whose frequency in the nucleotide sequence coded protein and order.Carry out stdn with regard in these selection pressures each, (for example calculate the sequence motifs of expecting in the genome then, length is the sequence motifs of 2 to 8 Nucleotide) average frequency, method with this frequency and this actual frequency comparison is used to seek sequence motifs then, described sequence motifs is compared with the people's gene group, excessively occurs or occur deficiency in the HIV-1 genome.This method is described in people such as common unsettled temporary patent application number 60/808,420 and Robins, and (p.8370-74, its content is integrated with this paper by reference for Journal ofBacteriology, (2005) the 187th volumes.

[0133] based on the biology knowledge of above-described HIV reagent, the HIV genome may comprise one or more INS motifs.We expect that these motifs are not present in host's (being the people) gene with the content (the HIV genome has high A content) that quite is rich in A.Identify and studied 4,000 people's genes by using aforesaid method with A content suitable with HIV.Identified to compare in these people's genes and insufficient sequence motifs AGG occurred with the frequency of expection.Find that identical AGG motif all excessively occurs in genomic gag gene of HIV-1 and pol gene.In 48 AGG oligonucleotide sequences (as shown in fig. 1) in being present in the gag gene, surpass 2/3rds and be not on the permanent staff in the amino acid whose reading frame of sign indicating number, hint that these sequences cannot not be conservative on amino acid/protein level owing to the reason of selecting.Yet the change of the 3rd codon position is common in different HIV strain isolateds, is conservative especially in the 3rd position of codon even also find the AGG motif.In addition, find that also the AGG motif (finding to comprise all the AGG motif of 44 to 48 copies) excessively occur in more than 400 that have analyzed different HIV-1 strain systems.These results show that the AGG motif can be reverse selection in people's gene group (promptly among the host at HIV), yet are maintained and/or enrichment in the HIV gene.Generally speaking, these results show that the AGG motif can be the INS sequence.

[0134] Embodiment 2-identifies the AGG motif in other slow viruss

[0135] existence of research AGG motif in many kinds of slow viruss, described slow virus comprises simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and the equine infectious anemia virus (EIAV) of HIV-2, several strain system.Find that all these viruses have the frequency of AGG motif expection or that be higher than expection.Yet, find that human T-cell leukemia virus (HTLV-1) and human reverse transcript transposon LINE-1 do not have the frequency of AGG motif expection or that be higher than expection.

[0136] Embodiment 3-is used to identify the method for other INS sequences

[0137] the HIV genome may comprise the other INS sequence except the AGG motif.The AGG motif also may form the part of bigger INS sequence.Above-mentioned method about evaluation AGG motif is used in the HIV genome or in fact finds other INS sequence in the genome of any other virus (for example other slow viruss or other retrovirus).

[0138] The function of embodiment 4-AGG motif

[0139] do not change the effect that amino acid whose coding is tested the AGG motif by suddenling change one or more motifs.For example, the plasmid transfection (instantaneous or stably) that will comprise the HIV-1gag sequence (being regulated and control by HIV LTR separately) of wild-type or sudden change is gone into the same cell type, for example uses PCR in real time to measure the steady state levels of the gag mRNA of generation then.Whether this experiment will detect AGG sequence in the gag gene influences transcription rate, this mRNA transformation period to cytoplasmic transhipment or mRNA.Use all has one or more AGG sequences of having suddenlyd change in gag and pol zone construct repeats this identical experiment.

[0140] in order to observe the effect of AGG motif on protein level, uses the construct of the encoding sequence that comprises GAG-POL and green fluorescent protein (GFP).The fluorescence detection method that uses standard then for example fluorescent microscopy detects the cell that produces GAG-POL-GFP.Also can use flow cytometry and fluorescence activated cell sorting.Nucleic acid transfection cell with the GAG-POL-GFP (promptly having one or more constructs that are mutated into the AGG motif of non-AGG motif) of encoding wild type GAG-POL-GFP or sudden change.Be expected at there being the much higher GFP that is detected of level in the mutant nucleotide sequence cells transfected.

[0141] if should experiment produce expected result, will prove that (1) AGG motif is that INS sequence and (2) AGG INS sequence can be used for reducing the steady state levels of HIV mRNA in cell.

[0142] also will detect the TAT and the REV dependency of these constructs.Even expection also produces high-caliber GAG-POL RNA (may be high 70 to 130 times than wild-type construct) with AGG sudden change construct cells transfected under TAT and the non-existent situation of REV.Expection is with TAT and/or REV expression construct or comprise TAT and the GAG-POL construct cotransfection cell of REV encoding sequence further increases the level of GAG-POL RNA.

[0143] Embodiment 5-vaccine

[0144] up to now, the immunity that does not also have the anti-HIV of can giving of commercially available acquisition to attack.Exist many reasons to explain before and can not produce such vaccine.A factor that may cause difficulty in producing vaccine can be HIV long-term ability that keeps in cell.The protected immunity of avoiding antibody-mediated (but non-CD-8T is cell-mediated) of intracellular virus.Because it produces speed slowly in cell, it remains on the ability of hiding in the cell and its by the ability of cytogamy from cellular invasion to cell, it is for a long time hidden that HIV virus can keep in cell.

[0145] these character of HIV virus can influence the ability that produces effective vaccine unfriendly on a plurality of levels.On a level, based on the vaccine of HIV virus for example deactivation or attenuation HIV vaccine can enter and be retained in the host cell with for a long time, wild-type HIV virus is also like this.Therefore, because the finite time that the slow life cycle of virus and virus expose in the extracellular, immunity system can not produce by force to the protective immunity of attacking subsequently that is enough to provide the anti-HIV of use.On another level, because for example existence of AGG motif in employed nucleic acid construct of INS sequence, DNA can express the antigen of extremely low-level HIV coding.Generally speaking, the antigen of generation is many more, and immune response will be big more.Therefore, if produce low-level HIV antigen, anti-these antigenic immune responses of generation also will be very low.

[0146] may be used in or one or more AGG motifs of being used to produce in the viral nucleic acid of vaccine overcome these problems by sudden change, thereby produce more effective vaccine.For example, can produce wherein, also carry out sudden change to destroy the attenuation HIV vaccine of one or more AGG motifs except being changed to reduce its ability that causes disease.

[0147], can produce the attenuation HIV virus of AGG motif with sudden change in order to detect aforesaid method.HIV protein that these mutated viruses host cells infecteds of use cell culture system in vitro study, expression are encoded and the ability that produces new virion.Equally, use appropriate H IV animal models infected to test these mutated viruses and in the host, produce immunoreactive ability in vivo.

[0148] in addition, use SIV virus and the identical method of FIV virus test.Generation has the attenuation FIV and the SIV virus of the AGG motif of sudden change.Use the ability of these mutated viruses cells infecteds of cell culture system in vitro study.The virus of these sudden changes of test produces immunoreactive ability in to the host of SIV and/or FIV infection susceptible equally, in vivo.These SIV and FIV experiment will be provided for the useful model that HIV vaccine/HIV infects.In addition, the generation of anti-SIV vaccine and test in the ape and monkey species, the generation of anti-FIV vaccine and test itself are useful in the cat kind.The immune composition of the provide protection that anti-SIV attacks can be provided in the ape and monkey species and/or can in the cat kind, provide anti-FIV provide protection immune composition within the scope of the invention.

[0149] Embodiment 6-HIV hides and the model of the effect of AGG motif in hiding

[0150] in the experiment similar, wild-type and mutant gag-pol construct stable transfection are gone into cell to above-mentioned experiment.Because having the clone of the integration copy of wild-type AGG, the activity of AGG sequence, expection will produce low-level GAG and POL RNA and protein.Change the phenomenon of (stochastic phenotypicswitching) owing to it is believed that the phenotype at random that participates in hiding, expect that also some clones will not produce any GAG-POL mRNA or protein.These clones will provide the cell culture model of hiding.

[0151] distinguishes with the clone who does not express GAG-POL for these being expressed the clone of low-level GAG-POL, can use the GAG-POL-GFP expression construct.Therefore, can detect express the cell of GAG-POL-GFP, and cell sorting art (for example fluorescence activated cell sorting or FACS) is used to separate this two kinds of cells, promptly express and do not express the cell of GAG-POL-GFP by fluorescence detection.Also may test these clones and in fact comprise the copy of the integration of the proteic nucleic acid of coding GAG-POL-GFP to confirm them.

[0152] transformation model of phenotype at random of hiding according to HIV, we expect that the percentage ratio of non-cloning by expression will begin the expression of GAG-POL-GFP on some points.Can measure frequency/rate that the GAG-POL gene opens and the environmental variance that influences this conversion then.The shape of hiding makes virus have more reactive reagent (for example proteinase inhibitor, reverse transcriptase inhibitors, DNA disrupting agent and other reagent) to treatment thereby such model is used for identify reversing HIV.

[0153] this model also can be used for studying the influence of AGG motif to hiding.Expection, under REV and the non-existent situation of TAT, the AGG sequence can play a role to reduce the steady state levels of HIV RNA.The AGG motif also may be able to be eliminated the generation of HIV RNA in the lump fully, as taking place in latent period.In fact, the AGG motif may be responsible for hiding of HIV or important factor wherein.

[0154] Conjugated protein and the reagent of embodiment 7-AGG

[0155] propose, the INS sequence can be by working in conjunction with cell protein, described albumen thereby to play a role to produce for transcribing be the chromatin (referring to people such as Schneider) of non-activity.In this research, in the gene of 4,000 screenings, identify 9 cytogenes with the same high AGG frequency with HIV.Therefore cell AGG is conjugated protein may be present in people's cell.In order to determine whether such albumen exists,, clean the T cell extract by AGG oligonucleotide post with rival people DNA if exist then separate described albumen.If identified such albumen, determine this proteinic aminoacid sequence and this proteinic gene of identification code.

[0156] if identify, such AGG is conjugated protein or that simulate described proteic effect (promptly by with the combining of AGG), and reagent can be used for inducing hiding of virus.If this wishes in some cases---can induce by the processing of using reagent and hide, its any consequence that may in the host who infects HIV be infected is reduced to bottom line or eliminates fully.Can expect that also such albumen or reagent have only minimum side effect to the host, because most of host gene has the AGG frequency than low 100 times of the AGG frequency of HIV.

[0157] in addition, if identified that such AGG is conjugated protein, may screen blocking-up or reduce described albumen to the bonded avidity of AGG or destroy AGG in conjunction with active reagent.This type of reagent can be used for reversing hiding in the turnover of HIV virus.This type of effect is wished in some cases, for example wishes under the ability situation that increases other drug and/or vaccine elimination HIV infection.

[0158] reagent and blocking-up, the protein-bonded avidity of minimizing AGG of conjugated protein, the simulation AGG protein-bonded effect of AGG or the effect of destroying its active reagent can be used the method for describing to detect herein and also can for example detect in the animal model of SIV and/or FIV at HIV.

[0159] Embodiment 8 the systematicness of HIV-1 genome and people's gene group relatively in mirror Fixed multiple Nucleotide motif

[0160] in the present embodiment, identify the multiple Nucleotide motif that in the nuclear restriction, plays origin cause of formation effect under a cloud in the systematicness of HIV-1 genome and people's gene group in relatively.Identified the short motif AGG that in people's gene group and HIV-1 genome, has the maximum difference performance between the encoding gene.The method of the application of the invention is carried out this evaluation.Described method has been identified the motif of showing significant difference in many performances between HIV-1 genome and people's encoding gene.In order to separate its contribution to expression level in the controlled experiment, the result who provides in the present embodiment concentrates on single motif of planting.Modify the codon optimization form of Gag, produce synonym and change with on the number that reduces the AGG generation.Produce two kinds of plasmids, a kind of sub-optimization of original password (CO) sequence with Gag, another kind has motif optimization (MO) sequence of the AGG that has remarkable minimizing.Described construct is transfected into human epithelial cell system (293 cell), and the expression of the expression ratio MO sequence of demonstration Gag is high by 70%.Also two sequences with Gag are prepared into injectable mouse vaccine to test two kinds of difference antibody responses between the construct.The mouse of the vaccine of use MO form has 4.5 times big anti-Gag antibody response after 4 weeks.The 4th week using DNA to strengthen and carrying out reading the second time in the 6th week, gap continues to strengthen the difference between MO and the CO vaccine.

[0161] method of the present invention (above-mentioned Robins-Krasnitz method) finds not rely on the short Nucleotide motif of amino acid whose order and codon selection in the coding region of people's gene group.Codon selects to be defined as the gross score that is illustrated in each password that uses in the given gene.The result of Robins-Krasnitz method is that a group length is 2 to 7 bases, occurs definite Nucleotide motif not enough and that excessively occur in the coding region of people's gene group.These basic sequences and HIV genome are compared.The first step of Robins-Krasnitz method is to produce and people's gene group background sequence relatively.This background is the completely random form from the encoding sequence of the people's gene group of the constraint that selected by amino acid order in each gene and codon.Can design the Monte Carlo program of the order of each intragenic each amino acid whose codon of randomly changing.Table 1 is illustrational example.

Table 1: the example of reorganization method

M L 1 L 2 H 1 L 3 H 2 L 4 H 3 ST ATG?CTA?CTG?CAT?TTA?CAT?CTG?CTT?TAG

[0162] method of acquisition maximum entropy distribution (MED) comprises one group randomization iteration (iteration).In the triplet of each amino acid whose Nucleotide of coding, change their order own randomly.This is to have 8 short proteic illustrative examples of amino acid whose simulation.Reorganization method (shuffling procedure) randomly changing L ₁, L ₂, L ₃And L ₄Order and change H respectively ₁, H ₂And H ₃Order.Each iteration produces new sequence.For present embodiment, there are 12 kinds of different combinations for leucine, there are 3 combinations for Histidine, thereby produce 36 unique sequences.In the reorganization method, they are weighted so that obtain MED in a large amount of multiple scopes.

[0163] above-mentioned reorganization method has produced one group randomized sequence.From these sequences, obtain probability distribution.As long as the generation number of each motif of finding in total sequence set is moderately big, can be by estimating that according to its mark in the group of all motifs the probability of given motif forms probability distribution.

[0164] after carrying out the reorganization method, determines two distributions, the true distribution of from actual sequence, finding and as the alternate maximum entropy distribution (MED) of background.The information theory standard is with acting on the method for selecting existing deficiency and the excessive motif that occurs.For the difference between true distribution and the MED forms the motif that maximum information bytes are provided is the first selection motif.Use information theory to have the desirable features of importing all results with identical unit, byte number.The comparison that this allows the motif of different lengths and insufficient basic sequence excessively occurs or occur.The formula that is used to calculate the motif of the maximum information bytes of contribution between two distributions is Kullback-Leibler distance or relative entropy.Calculate the relative entropy contribution of each motif, select maximum value.

[0165] in identifying sequence performance the most not enough or show over-drastic motif after, select the next not enough and next motif that excessively occurs that occurs.Yet, once can not obtain to have the motif of next maximum relative entropy simply.This is because motif is an eclipsed, the therefore not enough or excessively distribution of the every other motif of appearance influence of the appearance of given motif.The example of CpG is for example understood this point.In the people's gene group, dinucleotides motif CG will have maximum relative entropy.Yet, comprise CG and drop in the relative entropy motif of preceding 50 maximums as all 8 tripolymers of subgroup.This only is the artefact at the selection of CG.Recomputating relative entropy, need remove distribution from the CG of MED with before finding next motif.If basic sequence is called w, readjust all motifs that comprise w according to identical amount, the MED that readjusts like this has and the true identical distribution that distributes for w.This forces the relative entropy of w to become 0, simultaneously, eliminates the contribution from the w of every other motif.This selection of readjusting reduces the total relative entropy between the distribution singlely.

[0166] repeat described method, like this can be by the distribution of once removing a kind of motif from relative entropy of readjusting of MED.Then, select a kind of motif down.Along with the continuation of the described method of iteration, find other motif, until as determined by the genome of relatively reorganization, the motif with maximum residual relative entropy statistically significantly till.

[0167] start from one group 100 in the people's gene group performance the most not enough or show over-drastic basic sequence, after considering total " A " content, the method that provides has herein been identified the motif that has maximum density difference between HIV genome and people's gene group.Motif is defined in the group of people's gene with " A " content in 1% average HIV " A " content.Then with the density rate in the HIV genome divided by the density in the people coding region.If people's density is greater than the density of HIV, then with the described amount of its replacement reciprocal.Motif with maximum density ratio is the prediction for the isolating origin cause of formation signal of the nuclear of HIVmRNA.

[0168] considers the preferences of Nucleotide, find that the extremely insufficient AGG triplet of performance has high frequency in HIV in the coding region of people's gene group.The objective of the invention is will increase protein expression by encode the again ORF of HIV of the frequency that reduces motif AGG.

[0169] for this research, experimental test concentrates on the Gag gene.Remove encode the again codon sequence optimized (being called Adarc-Gag) of Gag of all AGG by systematicness like this, so that very rare codon is modified and do not imported to described aminoacid sequence.The result is RK-Gag.

[0170] first step is to determine that RK-Gag compares the expression that whether has increase with codon optimization form Adarc-Gag.Because the sub-optimization of partial password has been cancelled in the modification among the RK-Gag, thereby the minimizing of expection protein expression level, unless motif AGG significantly suppresses processing or the transhipment of mRNA.For expression level relatively, a kind of in-vitro transfection people 293 cells with two kinds of Gag in multi-form.Measure protein level, RK-Gag is than the optimized Adarc-Gag of codon high by 70% (Fig. 2).

[0171] for the growth of the almost twice of testing expression to immunoreactive influence, produce dna vaccination from each sequence.These dna vaccinations are injected into the back leg muscle of Balb/C mouse, after 4 weeks, provide booster shots then.On the 4th week and the 6th time-of-week point, measure anti-Gag antibody titers (about detailed content referring to method) by anti-P24ELISA.The result sees Fig. 3.In mouse model, 70% increase of vivoexpression is converted into the difference of the humoral immune reaction that becomes to surpass 5 times.

The Gag gene encode again to reduce the immune response that has improved significantly in the mouse model the HIV dna vaccination that occurs in of single triplet.Than rarer in mouse, so expected results is in the people even more remarkable in people's encoding sequence for this short motif.Need one group of step in order to activated vaccine clinically, described step comprises the ability of encodes equally again ENV ORF and its induction of immunity reaction of test.Another step can be sought the neutralizing antibody reaction in the primate.The purpose of this work provides: compare with the existing password optimization scheme, the HIV ORF that encodes again can improve performance and immunoreactive evidence greatly.In addition, the application of method of the present invention is the effective ways that produce one group the motif that should be integrated into re-encoding method.The systematicness of other motifs of determining by method of the present invention comprises to have improves the potentiality that roll up of showing in the present embodiment.

Claims (84)

1. one or more AGG sequences are changed over the viral nucleic acid of non-AGG sequence thereby having carried out suddenlys change.

2. the viral nucleic acid of claim 1, wherein said virus is slow virus.

3. the viral nucleic acid of claim 1, wherein said virus is HIV.

4. comprise the HIV nucleic acid that causes one or more AGG sequences to change over the sudden change of one or more non-AGG sequences.

5. comprise the viral nucleic acid that the one or more AGG sequences that cause in gag, pol or the env gene change over one or more sudden changes of non-AGG sequence.

6. comprise the HIV nucleic acid that the one or more AGG sequences that cause in gag, pol or the env gene change over one or more sudden changes of non-AGG sequence.

7. has the HIV nucleic acid that in the gag gene, has the sequence that is less than 47 AGG sequences.

8. has the HIV nucleic acid that in the gag gene, has the sequence that is less than about 45 AGG sequences.

9. has the HIV nucleic acid that in the gag gene, has the sequence that is less than about 43 AGG sequences.

10. has the HIV nucleic acid that in the gag gene, has the sequence that is less than about 41 AGG sequences.

11. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 39 AGG sequences.

12. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 37 AGG sequences.

13. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 35 AGG sequences.

14. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 33 AGG sequences.

15. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 31 AGG sequences.

16. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 29 AGG sequences.

17. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 27 AGG sequences.

18. have the HIV nucleic acid that in the gag gene, has the sequence that is less than about 25 AGG sequences.

19. have the HIV nucleic acid that in the gag gene, has the sequence that is less than 25 AGG sequences.

20. comprise the HIV nucleic acid of the AGG sudden change that shows among one or more Fig. 1.

21. be used to produce the method for the mutated viruses nucleic acid of the AGG sequence with one or more sudden changes, described method comprises: the viral nucleic acid that comprises one or more AGG sequences is provided and one or more AGG series jumps are become non-AGG sequence.

22. the method for claim 21, if wherein one or more AGG sequences are present in the zone of viral nucleic acid of coded protein, select non-AGG sequence that sudden change AGG sequence produces so so that it does not change the aminoacid sequence by described viral nucleic acid encoded protein matter.

23. the method for claim 21, if wherein one or more AGG sequences are present in the zone of viral nucleic acid of coded protein, select non-AGG sequence that sudden change AGG sequence produces so so that it does not change structure, function or immunogenicity by described viral nucleic acid encoded protein matter.

24. the method for claim 21, wherein make the step of carrying out one or more AGG sequences are changed over non-AGG sequence with the following method, described method is selected from the mutagenesis of mutagenesis, positive microbiotic back-and-forth method, single restriction site null method, deoxyuridine integration method, phosphorothioate integration method and the PCR-based of site-directed mutagenesis, oligonucleotide guidance.

25. the method for claim 21, wherein said viral nucleic acid comprises the whole genome of described virus.

26. the method for claim 21, wherein said viral nucleic acid comprises virus genomic part, and this part is enough to instruct the generation of virion in host cell.

27. the method for claim 21, wherein said viral nucleic acid comprises virus genomic part, and this part is enough to instruct the expression of at least a virus protein.

At least a virus protein or its fragment, derivative, variant or homologue 28. the method for claim 21, wherein said viral nucleic acid are encoded.

29. the method for claim 28, at least a virus protein and its fragment, derivative and the homologue that is selected from GAG, POL, ENV of wherein said viral nucleic acid coding.

30. having carried out suddenlys change changes over one or more AGG sequences the genomic mutated viruses of non-AGG sequence thereby have.

31. be not the recombinant virus of slow virus, having carried out suddenlys change changes over one or more AGG sequences the slow virus nucleotide sequence of non-AGG sequence thereby wherein said recombinant virus comprises.

32. having carried out suddenlys change changes over one or more AGG sequences the virus vaccines of the nucleic acid sequence of non-AGG sequence thereby comprise.

33. having carried out suddenlys change changes over one or more AGG sequences the HIV virus vaccines of the HIV nucleotide sequence of non-AGG sequence thereby comprise.

34. comprise the virus vaccines that has than the nucleic acid sequence of the AGG AGG still less of wild-type virus.

35. comprise the HIV vaccine that has than the HIV nucleotide sequence of the AGG AGG still less of wild-type HIV.

36. can be than the corresponding wild C-type virus C virus vaccines of highland marking protein more, wherein said virus vaccines comprises the nucleotide sequence that has than wild-type virus nucleotide sequence AGG sequence still less.

37. can be than the corresponding wild type HIV virus HIV vaccine of highland marking protein more, wherein said HIV vaccine comprises the nucleotide sequence that has than wild-type HIV nucleic acid sequence AGG sequence still less.

38. comprise by expressing the proteinic virus vaccines that viral nucleic acid produces, one or more AGG sequences changed over non-AGG sequence thereby described viral nucleic acid has carried out sudden change.

39. the virus vaccines of claim 38, wherein external in host cell marking protein.

40. the virus vaccines of claim 38 wherein uses acellular in-vitro transcription and translation system to express described protein.

41. comprise proteinic HIV vaccine, one or more AGG sequences changed over non-AGG sequence thereby described HIV nucleic acid has carried out sudden change from the HIV expression of nucleic acid.

42. the HIV vaccine of claim 41 is wherein expressed described protein external in host cell.

43. the HIV virus vaccines of claim 41 wherein uses acellular in-vitro transcription and translation system to express described protein.

44. comprise each the vaccine and the immunogenic composition of other components of claim 32-43, described other components are selected from pharmaceutically acceptable thinner, carrier, vehicle and adjuvant.

45. be used for immune experimenter with antiviral method, described method comprises the claim 32,34,36 and each vaccine or the immunogenic composition of 38-40 of described experimenter being used significant quantity.

46. be used for the method that immune experimenter attacks with anti-HIV, described method comprises the claim 33,35,37 and each vaccine or the immunogenic composition of 39-41 of described experimenter being used significant quantity.

47. be used for immune experimenter with antiviral method, described method comprises the nucleic acid of described experimenter being used the coding virus of significant quantity, thereby described nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.

48. be used for the method for immune experimenter with anti-HIV, described method comprises the nucleic acid of described experimenter being used the coding human immunodeficiency virus of significant quantity, thereby described nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.

49. be used for immune experimenter with antiviral method, described method comprises the virus protein from the viral nucleic acid expression of described experimenter being used significant quantity, thereby described viral nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.

50. be used for the method for immune experimenter with anti-HIV, described method comprises the protein from the HIV expression of nucleic acid of described experimenter being used significant quantity, thereby described HIV nucleic acid has carried out sudden change one or more AGG sequences is changed over non-AGG sequence.

51. be used for the method for optimization virus protein in host's generation, described method comprises

(a) provide (i) host cell, (ii) from nucleotide sequence of genomic at least a portion of host cell and (iii) nucleic acid sequence;

(b) evaluation is one or more compares with the stochastic distribution of Nucleotide, occurs nucleotide sequence motif not enough or that excessively occur in host sequences in host sequences, and wherein each motif length is about 6 Nucleotide;

(c) determine whether described virus sequence comprises one or more being accredited as and motif not enough or that excessively occur occurs in (b);

(d) change described virus sequence with number that increases the insufficient sequence of appearance or the number that reduces the sequence that excessively occurs, or carry out both simultaneously, with the proteinic generation of optimization in the host.

Be used for the nucleic acid sequence of vaccine generation so that described nucleic acid sequence obtains change 52. be used for optimization, thus the method for the generation of the virus protein that acquisition increases, and described method comprises:

(a) provide (i) host cell, (ii) from nucleotide sequence of genomic at least a portion of host cell and (iii) nucleic acid sequence;

(b) evaluation is one or more compares with the stochastic distribution of Nucleotide, occurs nucleotide sequence motif not enough or that excessively occur in host sequences in host sequences, and wherein each motif length is about 12 Nucleotide of about 6-;

(c) determine whether described virus sequence comprises one or more being accredited as and motif not enough or that excessively occur occurs in (b);

(d) change the number that described virus sequence the number of insufficient sequence occurs with increase or reduces the sequence that excessively occurs, or carry out both simultaneously, with the described nucleic acid sequence of optimization, thus the protein output that acquisition increases in the host.

53. the method for claim 51 or 52, the length of wherein said part is 50kb at least.

54. the method for claim 51 or 52, the length of wherein said part is 100kb at least.

55. the method for claim 51 or 52, wherein said part are one or more est sequences.

56. the method for claim 51 or 52, wherein said optimized nucleic acid sequence is more effectively expressed than not optimized viral nucleic acid in host cell.

57. the method for claim 51 or 52 does not wherein change after the variation of gained aminoacid sequence in (d) by described virus sequence coding.

58. the method for claim 51 or 52, wherein said host cell is an eukaryotic cell.

59. the method for claim 51 or 52, wherein said host cell is a prokaryotic cell prokaryocyte.

60. the method for claim 51 or 52, wherein said host cell is a bacterial cell.

61. the method for claim 51 or 52, wherein said host cell is an insect cell.

62. the method for claim 51 or 52, wherein said host cell is a mammalian cell.

63. the method for claim 51 or 52, wherein said host cell are people's cells.

64. the method for claim 51 or 52, wherein said host cell is a primates zooblast.

65. the method for claim 51 or 52, wherein said host cell is a Chinese hamster ovary celI.

66. the method for claim 51 or 52, wherein said host cell is a mouse cell.

67. the method for claim 51 or 52, wherein said host cell is a yeast cell.

68. the method for claim 51 or 52, wherein said host cell are the goat cells.

69. the method for claim 51 or 52, wherein said host cell are the sheep cells.

70. the method for claim 51 or 52, wherein said host cell are the birds cells.

71. the method for claim 51 or 52, wherein said host cell is a chicken cell.

72. the method for claim 51 or 52, wherein said host cell is in transgenic animal.

73. the method for claim 51 or 52, wherein said evaluation comprises:

A) provide the genome sequence of host cell,

B) provide randomized background genome, its aminoacids content and codon with each gene identical with host genome is selected, but is at random,

C) carry out iterative method to identify that in the genome sequence of host cell length is the oligonucleotide sequence of about 7 Nucleotide of about 2-, described oligonucleotide sequence is represented following sequence, promptly this sequence is at utmost facilitated the difference between background genome and the host cell gene group, and wherein said method comprises step:

I) select the oligonucleotide sequence the most significantly host cell gene group and background genome are separated,

Ii) adjusting background probability once more distributes to get rid of because the difference that the oligonucleotide of selecting in the step (i) causes;

Iii) repeating step (i) is until the distribution of background distributions near the host, thereby identifies and compare with the stochastic distribution of Nucleotide, occurs nucleotide sequence motif not enough or that excessively occur in host sequences in host sequences.

74. increase the method for the output of purpose virus protein in host cell or the host living beings, wherein said protein is external source for the host, described method comprises the one or more Nucleotide in the described nucleic acid sequences to proteins of permutation encoding, described like this displacement produces in described nucleotide sequence and compares with stochastic distribution, the one or more sequence motifs that in described host's nucleotide sequence, excessively occur, wherein make and identify the fragment that excessively occurs with the following method in described host's nucleotide sequence, described method comprises

A) provide the genome sequence of described host cell,

B) provide randomized background genome, its aminoacids content and codon with each gene identical with described host cell gene group is selected, but is at random

C) carry out iterative method to identify that in the genome sequence of described host cell length is the oligonucleotide sequence of about 7 Nucleotide of about 2-, the such sequence of described oligonucleotide sequence representative, promptly this sequence is at utmost facilitated the difference between described background genome and the described host cell gene group, and wherein said method comprises step:

I) select the oligonucleotide sequence the most described host cell gene group and described background genome are separated

Ii) adjusting background probability once more distributes to get rid of because the difference that the oligonucleotide of selecting in the step (i) causes;

Iii) repeating step (i) until background distributions near described host's distribution, thereby identify and compare with the stochastic distribution of Nucleotide, nucleotide sequence fragment not enough or that excessively occur in described host sequences appears in described host sequences.

75. increase the method for the output of virus protein in host cell or the host living beings, wherein said protein is external source for the host, described method comprises the one or more Nucleotide in the nucleotide sequence of permutation encoding purpose virus protein, described like this displacement is removed in exogenous nucleic acid sequences with the stochastic distribution of Nucleotide and is compared, insufficient one or more sequence motifs appear in described host's nucleotide sequence, wherein make and identify with the following method or determine in described host's nucleotide sequence and insufficient motif occurs, described method comprises

A) provide the genome sequence of described host cell,

B) provide randomized background genome, its aminoacids content and codon with each gene identical with described host cell gene group is selected, but is at random

C) carry out iterative method to identify that in the genome of described host cell length is the oligonucleotide sequence of about 7 Nucleotide of about 2-, the such sequence of described sequence representative, promptly this sequence is at utmost facilitated the difference between described background genome and the described host cell gene group, and wherein said method comprises step:

I) select the oligonucleotide sequence the most described host cell gene group and described background genome are separated,

Adjust once more ii) that background probability distributes to get rid of because the difference that the oligonucleotide of selection causes in the step (i) and

Iii) repeating step (i) is until the distribution of background distributions near described host; Thereby identify and compare, nucleotide sequence fragment not enough or that in described host sequences, excessively occur in described host sequences, occurs with the stochastic distribution of Nucleotide.

76. be used to identify the generation that suppresses or stimulate viral RNA, virus protein or virion, perhaps suppress or stimulate the compositions and methods of hiding of virus, described method comprises,

(a) provide the control cells that comprises at least one nucleic acid sequence that contains at least one AGG motif and comprise the subject cell that at least one contains the nucleic acid sequence of at least one AGG motif that has been mutated into non-AGG sequence,

(b) described subject cell is contacted with one or more reagent with described control cells and

(c) identify at least a reagent, and in described subject cell, compare that described reagent suppresses or stimulate the proteinic generation of generation, slow virus of viral RNA, the generation of virion in described control cells, perhaps suppress or stimulate hiding of virus.

77. be used to identify the method for AGG wedding agent, described method comprises

(a) provide the nucleic acid that comprises at least one AGG motif,

(b) described nucleic acid is contacted with one or more reagent and

(c) identify at least a reagent in conjunction with described nucleic acid.

78. be used to identify the method for AGG motif wedding agent, described method comprises

(a) provide the contrast nucleic acid that comprises at least one AGG motif and comprise the nucleic acid that tried of at least one AGG motif that has been mutated into non-AGG sequence,

(b) with described tried that nucleic acid contacts with one or more reagent with described contrast nucleic acid and

(c) identify at least a in conjunction with described contrast nucleic acid but the described reagent that is tried nucleic acid of debond.

79. be used to identify in conjunction with the reagent of AGG motif method, described method comprises,

(a) provide the control cells that comprises at least one nucleic acid sequence that contains at least one AGG motif and comprise the subject cell that at least one contains the nucleic acid sequence of at least one AGG motif that has been mutated into non-AGG sequence,

(b) described subject cell is contacted with one or more reagent with described control cells and

(c) identify at least a reagent, compare with described subject cell that described reagent suppresses or stimulate the generation of viral RNA, the generation of virus protein, the generation of virion in described control cells, perhaps inhibition or stimulation virus hides.

80. the AGG motif wedding agent that the method for each of use claim 77-79 is identified.

81. suppress or stimulation AGG wedding agent and the bonded reagent that comprises the nucleic acid of at least one AGG motif.

82. be used to identify and suppress or stimulate AGG wedding agent and the bonded compositions and methods that comprises the nucleic acid of at least one AGG motif, described method to comprise,

(a) provide the nucleic acid that comprises at least one AGG motif,

(b) described nucleic acid is contacted with at least a second reagent with the AGG wedding agent and

(c) identify bonded second reagent that suppresses or stimulate AGG wedding agent and described nucleic acid.

83. claim 5,21,30,32,34,36,38,44,45,47,49,51,52,74,75,76 or 79 each method, wherein said virus is slow virus.

84. claim 5,21,30,32,34,36,38,44,45,47,49,51,52,74,75,76 or 79 each method, wherein said virus is HIV.

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