DK175613B1 - Virus vector encoding a glycoprotein from the virus responsible for AIDS, vaccine comprising the virus vector or glycoprotein, and antibody to the glycoprotein - Google Patents
Virus vector encoding a glycoprotein from the virus responsible for AIDS, vaccine comprising the virus vector or glycoprotein, and antibody to the glycoprotein Download PDFInfo
- Publication number
- DK175613B1 DK175613B1 DK198706417A DK641787A DK175613B1 DK 175613 B1 DK175613 B1 DK 175613B1 DK 198706417 A DK198706417 A DK 198706417A DK 641787 A DK641787 A DK 641787A DK 175613 B1 DK175613 B1 DK 175613B1
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- DK
- Denmark
- Prior art keywords
- virus
- glycoprotein
- sequence
- virus vector
- gene
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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Abstract
Description
DK 175613 B1 IDK 175613 B1 I
Den foreliggende opfindelse angår en vaccine beregnet til forebyggelse af AIDS. IThe present invention relates to a vaccine intended for the prevention of AIDS. IN
Erhvervet immundefektsyndrom (AIDS) er en viruslidelse, som har nået et betyde- IAcquired immunodeficiency syndrome (AIDS) is a viral disorder that has reached a significant level.
ligt omfang i Nordamerika, Europa og Centralafrika.similar scope in North America, Europe and Central Africa.
De seneste beregninger tyder på, at ca. 1 million amerikanere kan have været IThe latest calculations indicate that approx. 1 million Americans may have been you
udsat for AIDS-virus. De ramte personer udviser en alvorlig immundepression, og Iexposed to AIDS virus. The affected individuals exhibit a severe immune depression, and I
sygdommen er generelt dødelig. Ithe disease is generally fatal. IN
10 Overførsel af sygdommen sker oftest ved seksuel kontakt, selv om stiknarkomaner også udgør en højrisikogruppe; desuden er et stort antal personer blevet inficeret med dette virus efter at have modtaget kontamineret blod eller blodprodukter.10 The transmission of the disease is most often through sexual contact, although drug addicts also constitute a high-risk group; moreover, a large number of people have been infected with this virus after receiving contaminated blood or blood products.
Det agens, der forårsager denne lidelse, er et retrovirus. Adskillige lidelser hos dyr 15 er blevet tilskrevet retrovira, men man har først for nylig kunnet beskrive retrovira, der rammer mennesker.The agent that causes this disorder is a retrovirus. Several disorders in animals 15 have been attributed to retroviruses, but it has only recently been possible to describe retroviruses affecting humans.
Medens retrovira fra humane T-celler (HTLV: human T leukemia virus) af type I og II har været angivet som agens for visse T-celleleukæmier hos voksne, er det re-20 trovirus, som er forbundet med lymphadenopatier (LAV-virus), og som også betegnes HTLV Ill-virus eller AIDS-relateret virus (ARV), nu almindeligt anerkendt som det ansvarlige agens for AIDS.While retroviruses from human T cells (HTLV: human T leukemia virus) of types I and II have been reported as agents for certain adult T cell leukemia, it is the retrovirus associated with lymphadenopathies (LAV virus). , and also referred to as HTLV III virus or AIDS-related virus (ARV), now widely recognized as the responsible agent for AIDS.
LAV-retrovirusgenomet er blevet karakteriseret særdeles grundigt (Wain-Hobson et I 25 al., 1985; Ratner et al., 1985; Muesing et al., 1985; Sanchez-Pescador et al., I 1985), og informationer om sekvensen viser en nær forbindelse med lentivirus- I gruppen. Lentivira, hvis prototype er Visna-fårevirus, er agentier for sygdomme I med meget langsom progression, og som typisk har en lang inkubationstid. LAV- og Visna-virus har mange fælles træk, især med hensyn til deres tropisme for I 30 nervevæv.The LAV retroviral genome has been characterized very extensively (Wain-Hobson et al., 1985; Ratner et al., 1985; Muesing et al., 1985; Sanchez-Pescador et al., 1985) and sequence information shows a close connection with the lentivirus I group. Lentivira, whose prototype is Visna sheep virus, is the agent of disease I with very slow progression and typically has a long incubation period. LAV and Visna viruses have many common features, especially with regard to their tropism for I 30 nerve tissue.
Ved analogi med andre velkendte retrovira har de tre vigtigste dele af LAV- I genomet fået betegnelsen gag, pol og env. env-gensekvensen, der omfatter I gpl20- og gp-41-sekvensen, har de karakteristika, som kunne forventes for et I 35 glycoprotein med transmembrankappe, og identiteten af precursoren for env- I DK 175613 B1 H proteinet, gpl60, som udgøres af gpl20 og gp41, er blevet bekræftet ved direkte H sekventering af aminosyrerne.By analogy with other well-known retroviruses, the three most important parts of the LAV-I genome have been named gag, pole and env. The env gene sequence comprising the I gp120 and gp-41 sequence has the characteristics that might be expected for a transmembrane envelope glycoprotein and the identity of the precursor of the env1, gp160, which is constituted by the gp120 and gp41, have been confirmed by direct H sequencing of the amino acids.
Η I det følgende betegnes gp41 undertiden gp40 eller gp42.Η In the following, gp41 is sometimes referred to as gp40 or gp42.
H Antistoffer mod env-proteinet gpl60 og dets spaltningsprodukter gpl20 og gp41 H detekteres sædvanligvis i serum fra AIDS-patienter, og env-Qlycoproteinet udgør H hovedoverfladeantigenet fra AIDS-virus.H Antibodies to the env protein gpl60 and its cleavage products gpl20 and gp41 H are usually detected in serum from AIDS patients, and the env-Qlycoprotein H is the major surface antigen of the AIDS virus.
10 env-proteinet er således den mest lovende kandidat til udvikling af en vaccina- tionsstrategi, hvorfor opmærksomheden er blevet koncentreret om dette protein og om den sekvens, der koder derfor.Thus, the env protein is the most promising candidate for developing a vaccination strategy, which is why attention has been focused on this protein and the sequence encoding it.
Et stort antal forskergrupper har beskrevet ekspressionen af env-proteinet i bakte- 15 rier. Imidlertid kan fraværet af glycosylering og posttranslationel strukturering kompromittere den immunogene evne hos de materialer, der syntetiseres af sådanne mikroorganismer.A large number of research groups have described the expression of the env protein in bacteria. However, the absence of glycosylation and post-translational structuring may compromise the immunogenic ability of the materials synthesized by such microorganisms.
H Ifølge den foreliggende opfindelse foreslås det derfor, at man som ekspressions- 20 vektor for env-proteinet anvender en virusvektor, der tillader ekspression af proteinet i et miljø, som muliggør dets glycosylering og posttranslationelle restrukturering.H According to the present invention, it is therefore proposed to use as an expression vector for the env protein a viral vector which allows expression of the protein in an environment which enables its glycosylation and post-translational restructuring.
Den foreliggende opfindelse angår således en virusvektor, der er ejendommelig 25 ved, at den omfatter det hele eller en del af env-genet fra det virus, der er I ansvarligt for AIDS.Thus, the present invention relates to a viral vector which is characterized in that it comprises all or part of the env gene from the virus responsible for AIDS.
I Blandt de anvendelige virusvektorer skal især nævnes poxvirus, navnlig vac- ciniavirus (W).In particular, among the usable virus vectors are pox viruses, especially vaccinia virus (W).
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Vacciniavirus er et virus med dobbeltstrenget DNA, som i vid udstrækning er blevet benyttet over hele verden til bekæmpelse og udryddelse af kopper. De seneste tekniske udviklinger har gjort det muligt at udvikle dette virus som kloningsvektor, og rekombinante levende vira har gjort det muligt at udtrykke fremmede antigener 35 og endog opnå immunisering mod forskellige virus- eller parasitsygdomme.Vaccinia virus is a double-stranded DNA virus that has been widely used throughout the world for the control and eradication of smallpox. Recent technical developments have made it possible to develop this virus as a cloning vector, and recombinant living viruses have made it possible to express foreign antigens and even achieve immunization against various viral or parasitic diseases.
3 DK 175613 B13 DK 175613 B1
Flere forskergrupper har således for nylig påvist anvendelsen af denne type rekombinanter til ekspression af antigenet for influenza, hepatitis B og rabies-glycoproteinet til immunisering mod disse sygdomme (Smith et al., 1983; Panicali 5 et al., 1983; Kieny et al., 1984).Thus, several research groups have recently demonstrated the use of this type of recombinants to express the antigen for influenza, hepatitis B and the rabies glycoprotein for immunization against these diseases (Smith et al., 1983; Panicali 5 et al., 1983; Kieny et al. , 1984).
Ekspression af en sekvens, som koder for et fremmed protein, ved hjælp af vacciniavirus (VV) omfatter nødvendigvis to trin: 10 1) den kodende sekvens skal anbringes sammen med en VV-promotor og indsættes i et ikke-essentielt W-DNA-segment klonet i et hensigtsmæssigt bakterieplasmid; 2) de W-DNA-sekvenser, der befinder sig på begge sider af den kodende sekvens, skal muliggøre homologe rekombinationer in vivo mellem 15 plasmidet og virusgenomet; en dobbelt reciprok rekombination fører til en overførsel af DNA-indsætningen fra plasmidet til det virusgenom, hvori den propageres og udtrykkes (Panicali og Paoletti, 1982; Mackett et al., 1982;Expression of a sequence encoding a foreign protein by vaccinia virus (VV) necessarily involves two steps: 1) the coding sequence must be placed with a VV promoter and inserted into a non-essential W-DNA segment cloned into an appropriate bacterial plasmid; 2) the W-DNA sequences located on both sides of the coding sequence should allow for in vivo homologous recombinations between the plasmid and the virus genome; a double reciprocal recombination leads to a transfer of the DNA insert from the plasmid to the viral genome in which it is propagated and expressed (Panicali and Paoletti, 1982; Mackett et al., 1982;
Smith et al., 1983; Panicali et al., 1983).Smith et al., 1983; Panicali et al., 1983).
20 Naturligvis medfører anvendelsen af denne type vektor ofte en delvis deletion af virusvektorgenomet.Of course, the use of this type of vector often results in a partial deletion of the virus vector genome.
Den foreliggende opfindelse angår mere specifikt en virusvektor, der er ejendommelig ved, at den i det mindste omfatter 25 en del af genomet fra en virusvektor, et gen, som koder for ét af glycoproteinerne (gp) fra kappen på det virus, der er ansvarligt for AIDS, og de elementer, der sørger for ekspression af dette glycoprotein i celler.More specifically, the present invention relates to a viral vector, characterized in that it comprises at least a portion of the genome of a viral vector, a gene encoding one of the glycoproteins (gp) of the envelope of the virus responsible. for AIDS, and the elements that provide expression of this glycoprotein in cells.
30 i30 i
Opfindelsen angår også de rekombinante DNA'er, der svarer til disse virusvektorer.The invention also relates to the recombinant DNAs corresponding to these viral vectors.
Det skal bemærkes, at der findes 3 glycoproteiner (gp) fra kappen på det virus, der er ansvarligt for AIDS, hvilke proteiner betegnes ved hjælp af deres masse i kD, 35 nemlig gpl60, gpl20 og gp41; det første, gpl60, er faktisk precursor for de toIt should be noted that there are 3 glycoproteins (gp) from the envelope of the virus responsible for AIDS, which proteins are designated by their mass in kD, namely gp160, gp120 and gp41; The first, the GPL60, is actually the precursor of the two
I DK 175613 B1 II DK 175613 B1 I
I II I
I sidstnævnte proteiner. Disse betegnelser ligger endnu ikke fast, og gp41 betegnes IIn the latter proteins. These names are not yet fixed and gp41 is designated I
I undertiden gp40 eller gp42, men Forskellene i masse gør, at disse 3 glycoproteiner IIn sometimes gp40 or gp42, but the differences in mass make these 3 glycoproteins I
I let kan identificeres, uanset hvilken betegnelse de har. IYou can easily be identified by whatever designation they have. IN
I 5 Med "virus, der er ansvarligt for AIDS", betegnes især LAV-virus, HTLV Ill-virus II 5 "Virus responsible for AIDS" refers in particular to LAV virus, HTLV III virus I
I eller ARV samt eventuelle punktmutanter eller delvise deletioner af disse vira samt II or ARV as well as any point mutants or partial deletions of these viruses as well as
I beslægtede vira. IIn related viruses. IN
I Virusvektorerne i den del, der svarer til virusvektorgenomet (forskellig fra det IIn the Virus vectors in the part corresponding to the virus vector genome (different from the I
I 10 virus, der er ansvarligt for AIDS), kan fremstilles ud fra genomet fra et virus af en IIn 10 viruses responsible for AIDS) can be produced from the genome of a virus of an I
I hvilken som helst oprindelse. Det foretrækkes imidlertid at anvende en del af IIn any origin. However, it is preferred to use part of I
I genomet fra et poxvirus og især en del af vaccinia-genomet. IIn the genome of a pox virus and especially part of the vaccinia genome. IN
I De betingelser, der kræves til ekspression af et heterologt protein i vacciniavirus, IIn the conditions required for expression of a heterologous protein in vaccinia virus,
I 15 er beskrevet tidligere. II 15 has been described previously. IN
I Generelt skal det pågældende gen, fx env-genet, for at kunne udtrykkes være IIn General, in order for expression to be expressed, the gene in question must be I
I under kontrol af en vacciniagenpromotor, og denne promotor er almindeligvis IYou are under the control of a vaccinia gene promoter and this promoter is usually I
I promotoren fra 7,5K-vacciniaproteinet. Desuden skal den kodende sekvens klones i IIn the promoter of the 7.5K vaccinia protein. In addition, the coding sequence must be cloned into I
I 20 et ikke-essentielt vacciniagen, der eventuelt kan tjene som markørgen. I de fleste IIn a non-essential vaccinia gene that may serve as the marker gene. In most I
I tilfælde drejer det sig om TK-genet. IIn this case, it is the TK gene. IN
I Blandt de kappeglycoproteiner, som man ønsker at udtrykke, skal nævnes de 3 IAmong the envelope glycoproteins that one wishes to express are the 3 I
I ovenfor nævnte proteiner, dvs. gpl60, gp41 og gpl20. IIn the above mentioned proteins, i.e. gpl60, gp41 and gpl20. IN
I 25 II 25 I
I Generelt foretrækkes det at lade det komplette kappegen udtrykkes, dvs. env- IIn General, it is preferable to have the complete cutting gene expressed, i.e. env- I
I genet omfattende dette gens signalsekvens og transmembransekvens. IIn the gene comprising this gene's signal sequence and transmembrane sequence. IN
I De første forsøg, der er udført med en virusvektor, hvori det gen, som koder for IIn the first experiments performed with a virus vector, wherein the gene encoding I
I 30 det totale env-protein, er blevet klonet, har ført til forslag om at modificere dette IIn the total env protein, which has been cloned, has led to proposals to modify it
I gen for at forbedre ekspressionsprodukternes immunogenicitet. IIn gene to enhance the immunogenicity of expression products. IN
I Der er konstateret en betydelig udskillelse af eny-proteinet i kultursupernatanterne ISignificant secretion of the eny protein in the culture supernatants I has been observed
I (en udskillelse, der sker i kredsløbsvæskerne, formodentlig in vivo!. Dette kan II (a secretion that occurs in the circulatory fluids, presumably in vivo!)
I 35 skyldes en dårlig vedhæftning af proteinet til cellemembranen; det er desuden IIn 35 is due to poor attachment of the protein to the cell membrane; moreover, you are
5 DK 175613 B1 kendt, at antigenernes præsentation pi celleoverfladen er meget vigtig for induktionen af et immunrespons med vaccinia-systemet. Det foreslås derfor at modificere env-genet på en sådan måde, at glycoproteinets forankring til celle-membranen forbedres.It is known that the presentation of the antigens on the cell surface is very important for the induction of an immune response with the vaccinia system. Therefore, it is proposed to modify the env gene in such a way that the anchoring of the glycoprotein to the cell membrane is enhanced.
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Til dette formål kan env-genet modificeres i den del deraf, der koder for transmembranområdet, for at erstatte kodonen svarende til arginin med en kodon svarende til isoleucin.To this end, the env gene may be modified in the portion thereof encoding the transmembrane region to replace the argonine-like codon with a isoleucine-like codon.
10 Der er også mulighed for at forbedre forankringen ved at erstatte en v-proteinets transmembranområde med transmembranområdet fra et heterologt virus, fx transmembranområdet fra gp fra rabiesvirus, og/-eller ved at føje denne zone til env-proteinets transmembranområde.It is also possible to improve the anchorage by replacing the transmembrane region of a v protein with the transmembrane region of a heterologous virus, e.g., the transmembrane region of gp from rabies virus, and / or by adding this zone to the transmembrane region of the env protein.
15 Desuden kan det ske, at proteinet ikke er korrekt samlet efter ekspressionen deraf. Signalpeptidet er nemlig temmelig atypisk og kan hæmme proteinets fuldstændige sekretion. Af denne grund foreslås det at erstatte og/eller tilføje en signalsekvens, som stammer fra et heterologt virus, fx signalsekvensen fra gp fra rabiesvirus.In addition, the protein may not be properly assembled after its expression. The signal peptide is quite atypical and can inhibit the complete secretion of the protein. For this reason, it is proposed to replace and / or add a signal sequence derived from a heterologous virus, for example the signal sequence from gp from rabies virus.
20 Det ser endelig ud til, at det er gpl20 og ikke gpl60, som udskilles af cellerne. Det kan på den ene side tilvejebringe en lokkemad for immunsystemet og på den anden side, hvilket stemmer overens med de seneste data, fæstne sig til T4-cellerne, hvilket kan bevirke, at T4-cellerne inaktiveres, eller at de ser fremmede ud for andre T-celler.20 Finally, it appears that it is gp120 and not gp160 that is secreted by the cells. It may, on the one hand, provide a bait for the immune system and on the other, which is consistent with the latest data, attach to the T4 cells, which may cause the T4 cells to be inactivated or make them appear foreign to others. T cells.
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Det kan således være fordelagtigt at få et env-protein gpl20, som ikke kan blive udskilt. Dette udføres ved at modificere env-genet mellem de sekvenser, der koder for gpl20 og gp41, for at undertrykke de spaltningssteder for proteaserne, som befinder sig mellem gpl20 og gp41, især ved fjernelse af REKR-stedet.Thus, it may be advantageous to obtain an env protein gp120 which cannot be secreted. This is done by modifying the env gene between the sequences encoding gp120 and gp41 to suppress the cleavage sites of the proteases located between gp120 and gp41, especially by removing the REKR site.
30 I plasmiderne ifølge opfindelsen udføres der mindst én yderligere mutation i et sted svarende til en KKR-sekvens 8 aminosyrer nedstrøms for REKR-sekvensen. Det således vundne gpl60 bliver ikke længere spaltet.In the plasmids of the invention, at least one additional mutation is carried out at a site corresponding to a KKR sequence 8 amino acids downstream of the REKR sequence. The gpl60 thus obtained is no longer cleaved.
I DK 175613 B1In DK 175613 B1
I 6 II 6 I
I Vektorerne ifølge opfindelsen omfatter også en sekvens, der koder for gp41 uden IIn the vectors of the invention also includes a sequence encoding gp41 without I
I den sekvens, der svarer til det hydrofobe N.-terminale peptid deraf, hvorom det IIn the sequence corresponding to the hydrophobic N. terminal peptide thereof, the I
I antages, at det kan være ansvarligt for env-proteinets syncytialevne, dvs. virusets IYou assume that it may be responsible for the syncytial ability of the env protein, ie. virus I
I evne til at få cellerne til at fusionere og derved opnå en kæmpecelle eller syncy- IIn the ability to cause the cells to fuse and thereby achieve a giant cell or syncope
I 5 tium. IFor 5 hours. IN
I Den foreliggende opfindelse angår endelig virusvektorer, i hvilke de ekstracytoplas- IThe present invention finally relates to viral vectors in which the extracytoplasm I
I miske og intracytoplasmiske gpl60-regioner er fusioneret i fase efter deletion af I det hydrofobe C-terminale peptid, hvilket gør det muligt at opnå sekretion af 10 glycoproteinet.In micaceous and intracytoplasmic gp160 regions are fused in phase following deletion of the hydrophobic C-terminal peptide, allowing secretion of the glycoprotein.
I Generelt omfatter virusvektorerne ifølge opfindelsen en sekvens, der koder for ét I af følgende proteiner: I 15 - S - gpl20-gp40 - (tm) - hvor I S er et signalpeptid, I - gpl20 er glycoproteinet 120, I 20 - J angiver den del af forbindelsen mellem gpl20 og gp40, der I mangler spaltningssted for proteaserne, I - gp40 er glycoproteinet 40, og - tm er et transmembranpeptid, I 25 eller for et protein med strukturen I - S - gp40 - tm I eller I - S - gpl20 - tm I 30 I Signalpeptidet og transmembransekvensen kan stamme fra det virus, der er an- I svarligt for AIDS, eller være heterologe, især stamme fra rabies- eller VSV-virus eller fra et hvilket som helst virus med kappe.In general, the virus vectors of the invention comprise a sequence encoding one I of the following proteins: I 15 - S - gp120-gp40 - (tm) - where IS is a signal peptide, I - gp120 is the glycoprotein 120, part of the link between gp120 and gp40, which lacks a cleavage site for the proteases, I - gp40 is the glycoprotein 40, and - tm is a transmembrane peptide, I 25 or for a protein of structure I - S - gp40 - tm I or I - S - gp120 - tm I 30 The signal peptide and transmembrane sequence may be derived from the virus responsible for AIDS or may be heterologous, especially from rabies or VSV virus or from any envelope virus.
I 35 7 DK 175613 B1 gp40 kan eventuelt mangle sin hydrofobe N-terminale ende.Optionally, the hydrophobic N-terminal end may be lacking in B1 gp40.
Den foreliggende opfindelse angår især anvendelse af virusvektorer til fremstilling af glycoproteiner, der indkodes af env-genet fra LAV-virus, i cellekulturer. Det 5 drejer sig således i første omgang om pattedyrceller, som er blevet inficeret med én virusvektor ifølge opfindelsen, eller som kan indeholde tilsvarende rekombinant DNA; blandt disse celler kan især nævnes diploide humane celler, primære kulturer samt Vero-celler. Det er naturligvis muligt at anvende andre typer celler, hvilket fremgår af nedenstående eksempler.In particular, the present invention relates to the use of virus vectors to produce glycoproteins encoded by the env gene from LAV virus in cell cultures. Thus, it is initially concerned with mammalian cells that have been infected with one viral vector of the invention or which may contain corresponding recombinant DNA; These cells include, in particular, diploid human cells, primary cultures and Vero cells. Of course, it is possible to use other types of cells, as shown in the examples below.
1010
De således vundne glycoproteiner kan efter oprensning anvendes til fremstilling af vacciner.The glycoproteins thus obtained can, after purification, be used to prepare vaccines.
Det er også muligt at overveje direkte anvendelse af virusvektorerne ifølge op-15 findelsen til udførelse af vaccination, idet glycoproteinerne da produceres in situ og in vivo.It is also possible to consider direct use of the virus vectors of the invention for performing vaccination, since the glycoproteins are then produced in situ and in vivo.
Det er fordelagtigt at påtænke den samlede anvendelse af flere vaccinationsmidler, der administreres sammen eller separat, især de vaccinationsmidler, som svarer til 20 de vektorer, der separat udtrykker gpl20 og gp40, og som har gennemgået den ovenfor beskrevne modifikation. Fx kan det være en fordel sammen at anvende de vaccinationsmidler, der stammer fra vektorerne 1136 og 1138, som beskrives nedenfor.It is advantageous to contemplate the overall use of multiple vaccines administered together or separately, especially those vaccines corresponding to 20 of the vectors expressing gp120 and gp40 separately and which have undergone the modification described above. For example, it may be advantageous to use the vaccine agents derived from vectors 1136 and 1138 described below.
25 Den foreliggende opfindelse angår endelig også antistoffer mod ovennævnte glycoproteiner, hvilke antistoffer er vundet ved infektion af en levende organisme med en virusvektor som beskrevet ovenfor samt isolering af de inducerede antistoffer efter et bestemt tidsrum.Finally, the present invention also relates to antibodies to the above glycoproteins, which antibodies are obtained by infection of a living organism with a virus vector as described above, and isolation of the induced antibodies after a certain period of time.
30 De teknikker, der anvendes til fremstilling af glycoproteiner, celle-kulturerne og vaccinationsteknikkerne er identiske med dem, der for tiden anvendes med de kendte vacciner, og beskrives ikke i detaljer.The techniques used to prepare glycoproteins, the cell cultures and vaccination techniques are identical to those currently used with the known vaccines and are not described in detail.
Opfindelsen belyses nærmere ved nedenstående metoder og eksempler samt under 35 henvisning til tegningen, hvorThe invention is illustrated in more detail by the following methods and examples and with reference to the drawing, where
I DK 175613 B1 II DK 175613 B1 I
I 8 II 8 I
I fig. 1 viser virkningen af endo-F på de proteiner, der er syntetiseret af IIn FIG. 1 shows the effect of endo-F on the proteins synthesized by I
I rekombinanterne VVTGeLAV9-l og VVTGeLAV1132 og IIn the recombinants VVTGeLAV9-1 and VVTGeLAV1132 and I
I immunudfældet ved hjælp af et anti-LAV-serum. I figuren er IIn the immunoprecipitated by an anti-LAV serum. In the figure, you are
I molekylvægte angivet i kilodalton, og IIn molecular weights given in kilodaltons, and I
I 5 - P betegner cellebundfaldet, II 5 - P denotes the cell precipitate, I
I - S betegner supematanten, II - S denotes the supernatant, I
I - u betegner produkter vundet uden behandling, II - u denotes products won without treatment,
I - e betegner produkter vundet efter behandling med endo-F. II - e denotes products gained after treatment with endo-F. IN
I 10 Fig. 2 viser genkendelse af LAV-virusproteinerne ved hjælp af IIn FIG. Figure 2 shows recognition of the LAV virus proteins by means of I
serum fra mus vaccineret med rekombinanten WTGeLAV9-l. I denne Iserum from mice vaccinated with the recombinant WTGeLAV9-1. In this I
I figur betegner T det tilfælde, hvor det anvendte serum stammer fra IIn Figure, T denotes the case where the serum used is derived from I
I en AIDS-patient. Molekylvægte er angivet kilodalton. IIn an AIDS patient. Molecular weights are given kilodaltons. IN
I 15 Fig. 3 viser immunudfældning af proteiner syntetiseret af de rekombinante IIn FIG. Figure 3 shows immunoprecipitation of proteins synthesized by the recombinant I
vacciniavira, der bærer env-oenet. I denne figur er molekylvægte Ivaccinia virus carrying the env. In this figure, molecular weights are I
I angivet i kilodalton. IIn indicated in kilodaltons. IN
I Fig. 4 viser immunudfældning af proteiner syntetiseret af de rekombinante IIn FIG. Figure 4 shows immunoprecipitation of proteins synthesized by the recombinant I
I 20 vira VV.TG.eLAV 1135, 1136, 1137 og 1138. IIn 20 viruses VV.TG.eLAV 1135, 1136, 1137 and 1138. I
I Virus 1135 syntetiserer et gpl60, som ikke ses i kultursupernatanten. IIn Virus 1135, a gp160 synthesizes, which is not seen in the culture supernatant. IN
Med hensyn til viraene 1136 og 1138 producerer de proteinerne IAs for the viruses 1136 and 1138, they produce the proteins I
henholdsvis gpl20 og gp40, der er forbundet med cellepelleten. Virus Igp120 and gp40, respectively, associated with the cell pellet. Virus I
I 1137 producerer et protein, der er en smule mindre end virus 1135 IIn 1137, a protein that is slightly less than virus produces 1135 I
I 25 med en molekylvægt, der svarer til den forventede. IIn 25 with a molecular weight that is similar to that expected. IN
I Fig. 5 viser strukturen af env-proteiner syntetiseret af de rekombinante IIn FIG. 5 shows the structure of env proteins synthesized by the recombinant I
I vira.In viruses.
I S signalpeptid 30 H intern hydrofob zone TM transmembran-forankringszone I t gpl20/gp40-spaltningssted I sekvens stammende fra rabies-glycoproteinet I 35 9 DK 175613 B1I S signal peptide 30 H internal hydrophobic zone TM transmembrane anchorage zone I t gp120 / gp40 cleavage site In sequence derived from the rabies glycoprotein I 35 9 DK 175613 B1
METODERMETHODS
Kloninger: Maniatis et al., 1982.Clones: Maniatis et al., 1982.
5 Enzymer: anvendt ifølge leverandørens anvisninger.5 Enzymes: used according to the supplier's instructions.
Lokaliseret mutaoenese: metode afledt fra Zoller og Smith, 1983.Localized mutaoenesis: method derived from Zoller and Smith, 1983.
Overførsel til vaccine: Kieny et al., 1984. Eneste forskel: de humane 143B-celler 10 erstatter LMTK'-cellerne.Transfer to vaccine: Kieny et al., 1984. Only difference: the human 143B cells 10 replace the LMTK 'cells.
Fremstilling af virus-stampræparation: Kimfri primære hønseceller inficeres med 0,01 pfu/celle i 4 dage ved en temperatur på 37°C (MEM-medium + 5% NCS).Preparation of Virus Stem Preparation: Germ-free primary chicken cells are infected with 0.01 pfu / cell for 4 days at a temperature of 37 ° C (MEM medium + 5% NCS).
15 Oprensning af virus: Der udføres oprensning af ovennævnte virus-stam-præpara-tion i 15 minutter ved 2500 omdr./minut (Rotor GSA Sorvall). Supernatanten sættes til side. Bundfaldet tages op i RSB-puffer (10 mM Tris-HCI, pH 7,4, 10 mM KCI, 1 mM MgCIV22) i 15 minutter ved 4°C. Der foretages en findeling i en homoge-nisator ("potter") og derefter centrifugering i 15 minutter ved 2500 omdr./minut.Purification of virus: Purification of the above virus strain preparation is performed for 15 minutes at 2500 rpm (Rotor GSA Sorvall). Set aside the supernatant. The precipitate is taken up in RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1 mM MgCIV22) for 15 minutes at 4 ° C. Triturate in a homogenizer ("pots") and then spin for 15 minutes at 2500 rpm.
20 Supernatanten sættes til den foregående, hvorefter der foretages endnu en findeling på samme måde.The supernatant is added to the previous one and then another comminution is made in the same way.
Alle supernatanterne anbringes på 10 ml 36% (vægt/volumen) saccha-rosepude (10 mM Tris, pH 8). Der udføres centrifugering i 2 timer ved 14.000 omdr./minut 25 (Rotor SW28, Beckman).All supernatants are applied to 10 ml of 36% (w / v) sucrose pad (10 mM Tris, pH 8). Centrifugation is performed for 2 hours at 14,000 rpm (Rotor SW28, Beckman).
Bundfaldet opsamles, adskilles og anbringes på en anden identisk pude. Bundfald nr. 2 optages i 5 ml PBS og anbringes på en 20-40% saccharosegradient (10 mM Tris, pH 8) (samme rotor). Der udføres centrifugering i 45 minutter ved 12.000 30 omdr./minut.The precipitate is collected, separated and placed on another identical pad. Sediment # 2 is taken up in 5 ml of PBS and placed on a 20-40% sucrose gradient (10 mM Tris, pH 8) (same rotor). Centrifugation is performed for 45 minutes at 12,000 30 rpm.
Virusbåndet isoleres. Det udfældes ved centrifugering i 1 time ved 20.000 omdr./minut. Bundfaldet tages op i 10 mM Tris, pH 8.The virus band is isolated. It is precipitated by centrifugation for 1 hour at 20,000 rpm. The precipitate is taken up in 10 mM Tris, pH 8.
3535
I DK 175613 B1 II DK 175613 B1 I
I io II io I
I Immunudfældninoer IIn Immunoprecipitating I
I Der udføres infektion af BHK-21-celler (skåle med en diameter på 3 cm, 106 celler II Infection of BHK-21 cells (3 cm diameter dishes, 106 cells I) is performed
I pr. skål, dyrket i G-MEM + 10% FCS) med 0,2 pfu/-celle i 18 timer. Mediet dekan- II per dish, grown in G-MEM + 10% FCS) at 0.2 pfu / cell for 18 hours. The medium dean- I
I 5 teres og erstattes med 1 ml medium uden methionin og 10 μ! 35S-methionin II 5 teres and replace with 1 ml medium without methionine and 10 μl! 35S-methionine I
I (Amersham) pr. skål. II (Amersham) pr. Cheers. IN
I Efter 2 timer tilsættes et overskud af ikke-radioaktivt methionin. IAfter 2 hours, an excess of non-radioactive methionine is added. IN
Ved afslutningen af mærkningen udføres skrabning af de inficerede celler, centri- IAt the end of the labeling, scraping of the infected cells, centrifugal, is performed
I 10 fugering i 1 minut i en Eppendorf-centrifuge, separation af supernatant- og bund- IFor 10 minutes for 1 minute in an Eppendorf centrifuge, separation of supernatant and bottom I
I faidsfraktionerne, vask af bundfaldet én gang i PBS-puffer og derefter immunud- IIn the faids fractions, wash the precipitate once in PBS buffer and then immuno-I
I fældning og gelelektroforese (ifølge Lathe et al., 1980). IIn precipitation and gel electrophoresis (according to Lathe et al., 1980). IN
Endo-F-behandlina IEndo-F treatment I
I 15 II 15 I
I Efter immunudfældning af de mærkede proteiner med et serum fra en AIDS- II After immunoprecipitation of the labeled proteins with an AIDS-I serum
I patient optages protein-A/sepharose-fraktionen i IIn patient, the protein A / sepharose fraction is taken up in I
I 0,2 M Na-phosphat, pH 6,1 IIn 0.2 M Na phosphate, pH 6.1 I
I 20 0,05% SDS I0.05% SDS I
I 0,1% Nonidet P40 II 0.1% Nonidet P40 I
I 0,1% β-mercaptoethanol IIn 0.1% β-mercaptoethanol I
I 0,1% EDTA, pH 8 IIn 0.1% EDTA, pH 8 I
25 og koges i 5 minutter for at denaturere proteinerne. I25 and boiled for 5 minutes to denature the proteins. IN
I Der udføres inkubation i 20 timer ved 37°C med 4 enheder Endo-F pr. ml og IIncubation is performed for 20 hours at 37 ° C with 4 units of Endo-F per ml. ml and I
I derefter udfældning i 2 minutter i is med 1/5 volumen 100% TCA. Bundfaldet I vaskes 3 gange med 80% acetone, der tilsættes prøvepuffer, og det hele hældes I 30 på SDS-gel.Then precipitate for 2 minutes in ice with 1/5 volume of 100% TCA. The precipitate I is washed 3 times with 80% acetone, sample buffer is added and the whole is poured into 30 on SDS gel.
11 DK 175613 B111 DK 175613 B1
Bestemmelse af antistoffer ved ELISA-testDetermination of antibodies by ELISA assay
- LAV- LOW
Anvendelse af ELAVIA-testen (Pasteur-Diagnostic) med et andet fåre-antimuse-5 antistof bundet til peroxidase.Use of the ELAVIA (Pasteur-Diagnostic) test with another sheep anti-mouse antibody bound to peroxidase.
- Vaccine- Vaccine
Plader med 96 brønde (NUNC) med flad bund inkuberes i 18 timer ved 37°C med 107 pfu vildtype-vacciniavirus i carbonatpuffer. Pladerne mættes derefter med 10 0,01% gelatine. Musesera adsorberes derefter til pladerne, og resten af proceduren udføres som for LAV-ELISA.96-well flat bottom plates (NUNC) are incubated for 18 hours at 37 ° C with 107 pfu wild-type vaccinia virus in carbonate buffer. The plates are then saturated with 10 0.01% gelatin. Mouse sera are then adsorbed to the plates and the rest of the procedure is performed as for LAV-ELISA.
Læsninger ved 492 nM.Readings at 492 nM.
15 EKSEMPEL 1EXAMPLE 1
Konstruktion af hybride plasmiderConstruction of hybrid plasmids
De kombinerede størrelser af de forskellige elementer, der er nødvendige for 20 overførsel af den sekvens, der koder for env-genet, til VV-genomet og efterfølgende ekspression deraf, er i størrelsesordenen flere kb. Det blev derfor anset for nødvendigt at minimere størrelsen af det replikationsplasmid i E. coli. der blev anvendt til konstruktionsarbejdet, for at lette de nødvendige manipulationer.The combined sizes of the various elements required for transfer of the sequence encoding the env gene to the VV genome and subsequent expression thereof are on the order of several kb. Therefore, it was considered necessary to minimize the size of the replication plasmid in E. coli. used for the construction work, to facilitate the necessary manipulations.
25 HindIII-(Hin-J)-fragmentet fra VV-genomet indeholder hele genet for thymidinki-nase (TK), som allerede tidligere er blevet anvendt til udskiftning og rekombination af DNA indsat i VV-genomet (Mackett et al., 1982). Det skal bemærkes, at overførsel af en indsætning til TK-genet fra VV-genomet giver et virus uden TK, som kan selekteres. Det var nødvendigt først at fremstille et lille plasmid, som bar et 30 unikt Hindlll-sted, der kunne anvendes til indsætning af Hin-J-W-fragmentet.The HindIII (Hin-J) fragment from the VV genome contains the entire gene for thymidine kinase (TK), which has already been used previously for replacement and recombination of DNA inserted into the VV genome (Mackett et al., 1982). . It should be noted that transfer of an insert to the TK gene from the VV genome produces a virus that can be selected without TK. It was first necessary to produce a small plasmid carrying a unique HindIII site that could be used for insertion of the Hin-J-W fragment.
Desuden var det nødvendigt at eliminere de for plasmidet unødvendige restriktionssekvenser, således at de efterfølgende mani-pulationer blev mulige.In addition, it was necessary to eliminate the restriction sequences unnecessary to the plasmid, so that subsequent manipulations became possible.
Konstruktionen blev påbegyndt ud fra plasmidet pML2 (Lusky og Bot-chan, 1981), 35 som er en vektor, der er afledt fra plasmidet pBR322 ved spontan deletion, og iConstruction was started from plasmid pML2 (Lusky and Botchan, 1981), which is a vector derived from plasmid pBR322 by spontaneous deletion, and in
DK 175613 B1 IDK 175613 B1 I
12 I12 I
hvilket segmentet mellem nukleotiderne 1089 og 2491 er gået tabt. Først blev Iwhich segment between nucleotides 1089 and 2491 is lost. First you became
Pstl-sekvensen elimineret ved indsætning af Ahalll-Ahalll-fragmentet fra pUC8 IThe pst1 sequence is eliminated by inserting the AhallI-AhallI fragment from pUC8 I
(Vieira og Messing, 1982) mellem to AhaW-steder på pML2, idet 19 basepar blev I(Vieira and Messing, 1982) between two AhaW sites on pML2, with 19 base pairs becoming I
elimineret. Man anvendte "linker-tailing"-metoden (Lathe et al., 1984) for at ind- Ieliminated. The "linker-tailing" method (Lathe et al., 1984) was used to integrate
5 sætte en Hindlll-linker mellem Nrul- og EcoRI-stederne, der var behandlet med SI I5 establish a HindIII linker between the Nrul and EcoRI sites treated with SI I
fra dette plasmid, idet BamHI-stedet blev elimineret. Dette fører til et plasmid på Ifrom this plasmid, eliminating the BamHI site. This leads to a plasmid of I
2049 basepar, som bærer det funktionelle p-lactamasegen (der meddeler ampicil- I2049 base pairs carrying the functional β-lactamase gene (which communicates ampicill I)
linresistens), og som desuden omfatter et aktivt replikationsinitieringssted i E. coli Iline resistance), and further comprising an active replication initiation site in E. coli I
og et unikt Hindlll-restriktionssted. Iand a unique HindIII restriction site. IN
1° I1 ° I
Denne konstruktion blev betegnet pTGlH. IThis construct was designated pTG1H. IN
Hin-J-fragmentet fra VV-DNA, som bærer TK-genet, er tidligere blevet klonet i en IThe Hin-J fragment from the VV DNA carrying the TK gene has been previously cloned into an I
vektor stammende fra pBR327 (Drillien og Spehner, 1983). Dette fragment på 4,6 Ivector derived from pBR327 (Drillien and Spehner, 1983). This 4.6 I fragment
15 kb blev genklonet i Hindlll-stedet på pTGlH. Der blev selekteret en klon, i hvilken I15 kb was re-cloned into the HindIII site of pTG1H. A clone was selected in which I
TK-genet befinder sig distalt i forhold til det gen, som koder for ampicillinresistens. IThe TK gene is distal to the gene encoding ampicillin resistance. IN
Denne konstruktion, pTGlH-TK, blev anvendt som vektor i nedenstående forsøg. IThis construct, pTG1H-TK, was used as a vector in the experiments below. IN
20 Næste trin var at isolere en W-promotor, der kunne anvendes til at styre ekspres- IThe next step was to isolate a W promoter that could be used to control expression I
sionen af den sekvens, som koder for det gen, der skal udtrykkes. Promotoren fra Ithe sequence of the sequence encoding the gene to be expressed. The promoter from I
et tidligt gen, som koder for et protein på 7500 dalton (7,5 K), er tidligere med Ian early gene encoding a protein of 7500 daltons (7.5 K) is previously associated with I
held blevet anvendt til et identisk formål (Smith et al., 1983), og man fortsatte Ihave been successfully used for an identical purpose (Smith et al., 1983) and continued
derfor med isolering af dette segment. Itherefore with the isolation of this segment. IN
25 I25 I
7,5 K-genet sidder på et af de mindste Sall-fragmenter (Sal-S-fragment) fra VV- IThe 7.5 K gene sits on one of the smallest SalI (Sal-S) fragments from VV-I
genomet, type WR (Venkatasan et al., 1981). Da man fortrinsvis kloner de små Igenome, type WR (Venkatasan et al., 1981). Preferably, the small ones are cloned
fragmenter, bæres Sal-S-fragmentet af en stor del af de kloner, der fås ved direkte Ifragments, the Sal-S fragment is carried by a large proportion of the clones obtained by direct I
kloning af VV-DNA, type WR, spaltet med Sall i plasmidet pBR322. Dette fragment Icloning of VV DNA, type WR, digested with SalI in the plasmid pBR322. This fragment I
30 overføres til vektorbakteriofagen M13mp701 (se Kieny et al., 1983) ved Sall-spalt- I30 is transferred to the vector bacteriophage M13mp701 (see Kieny et al., 1983) by SalI
ning og religation, hvilket således giver fagen M13TGSal-S. Ining and religion, thus giving the subject M13TGSal-S. IN
I denne klon findes et Scal-sted i umiddelbar nærhed af initierings-ATG'et fra 7,5 IIn this clone, a Scal site is located in the immediate vicinity of the 7.5 I initialization ATG
K-genet. Nedstrøms for 7,5 K-genet findes unikke BamHI- og EcoRI-steder, der IK gene. Downstream of the 7.5 K gene are unique BamHI and EcoRI sites that I
35 stammer fra vektoren. BamHI- og Scal-stederne fusioneres ved hjælp af en Bglll- I35 are derived from the vector. The BamHI and Scal sites are fused with a BglII-I
13 DK 175613 B1 linker, 5’-CAGATCTG-3', efter at de ender, der er frembragt ved BamHI-spaltning, er blevet udfyldt med Klenow-fragmentet fra E. coli-polymerase. Ved denne proces elimineres Scal-stedet, mens BamHI-stedet gendannes, og det unikke EcoRI-sted forskubbes nedstrøms. Samtidig elimineres SalI-(AccI)-stedet nedstrøms,.og Sall-5 stedet opstrøms bliver således unikt.B1 linker, 5'-CAGATCTG-3 ', after the ends produced by Bam HI cleavage have been filled with the Klenow fragment of E. coli polymerase. In this process, the Scal site is eliminated while the BamHI site is restored and the unique EcoRI site is displaced downstream. At the same time, the SalI (AccI) site is eliminated downstream, and the SalI-5 site upstream thus becomes unique.
Denne konstruktion betegnes M13TG 7,5 K.This construction is referred to as M13TG 7.5 K.
Inde i Hin-J-fragmentet fra W-DNA findes Clal- og EcoRl-steder, som er adskilt af 10 ca. 30 basepar (Weir og Moss, 1983). 7,5 K-promotorfragmentet, som er til stede i M13TG 7,5 K, udskæres med Accl og EcoRI og klones mellem Clal- og EcoRI-stederne på pTGlH-TK, hvilket giver pTGlH-TK-P7,5K.Within the Hin-J fragment from W-DNA are Clal and EcoRl sites, which are separated by 10 30 base pairs (Weir and Moss, 1983). The 7.5 K promoter fragment present in M13TG 7.5 K is excised with Accl and EcoRI and cloned between the ClaI and EcoRI sites of pTG1H-TK to give pTG1H-TK-P7.5K.
Denne konstruktion fører til overførsel af de unikke BamHI- og EcoRl-steder fra 15 vektoren M13 umiddelbart nedstrøms for 7,5 K-promotorsekvensen. Disse unikke BamHI- og EcoRl-steder anvendes i følgende konstruktion.This construction leads to the transfer of the unique BamHI and EcoRl sites from the vector M13 immediately downstream of the 7.5 K promoter sequence. These unique BamHI and EcoRl sites are used in the following construction.
Polylinkersegmentet fra bakteriofagen M13TG131 (Kieny et al., 1983) udskæres med EcoRI og BglH og indsættes mellem EcoRI- og BamHI-stederne på plasmidet 20 pTGlH-TK-P7,5K, hvilket giver pTG186-po!y. I denne konstruktion er 10 restriktionssteder tilgængelige for kloning med et fremmed gen under kontrol af P7,5K.The polylinker segment of the bacteriophage M13TG131 (Kieny et al., 1983) is excised with EcoRI and BglH and inserted between the EcoRI and BamHI sites of the plasmid 20 pTG1H-TK-P7.5K to yield pTG186 poly. In this construct, 10 restriction sites are available for cloning with a foreign gene under the control of P7.5K.
EKSEMPEL 2 25EXAMPLE 2 25
Konstruktion af et plasmid, der bærer env-sekvensenConstruction of a plasmid carrying the env sequence
For at få en sekvens, der koder for env. foretages først samling af de to provirale segmenter klonet i plasmiderne PJ19-6 og PJ19-13.To get a sequence encoding env. first, the two proviral segments cloned into plasmids PJ19-6 and PJ19-13 are assembled.
3030
For at sikre en hensigtsmæssig translation af env-mRNA blev nukleotidsekvensen omkring det formodede translationsinitieringssted for env-genet modificeret for at blive tilpasset til consensussekvensen for eukaryote gener, hvilket udførtes ved en styret mutagenese med et oligonukleotid i nærheden af position 5767.To ensure appropriate translation of env mRNA, the nucleotide sequence around the putative translation initiation site of the env gene was modified to align with the consensus sequence of eukaryotic genes, which was performed by a controlled mutagenesis with an oligonucleotide at position 5767.
3535
I DK 175613 B1 II DK 175613 B1 I
I II I
I Plasmiderne P319-13 og P319-6 indeholder Hindlll-fragmenter fra LAV-provirus- IIn plasmids P319-13 and P319-6, HindIII fragments from LAV
I genomet omfattende nukleotiderne henholdsvis 1258-1698 og 1698-9173. IIn the genome, the nucleotides comprise 1258-1698 and 1698-9173, respectively. IN
Et EcoRl-Kpnl-fragment fra PJ19-13 (indeholdende initierings-ATG'et fra env) blev IAn EcoRl-KpnI fragment from PJ19-13 (containing the initiation ATG from env) was
5 indsat i fagen M13TG130, og der blev udført en styret mutagenese med et oligo- I5 inserted into the phage M13TG130 and a controlled mutagenesis with an oligopoly I was performed.
I nukleotid (med sekvensen 5'CTCTCA7TGTCACTGCAG-TCTGCTCTTTC) for at indføreIn nucleotide (with the sequence 5'CTCTCA7TGTCACTGCAG-TCTGCTCTTTC) to introduce
I et Pstl-sted opstrøms for env-translati-onsinitieringskodonen (position 5767) og for IAt a Pst I site upstream of the env translation initiation codon (position 5767) and for I
I at erstatte G i position -3 med A. Det muterede fragment blev derefter indført mel- I lem EcoRI- og KpnI-stederne på plasmidet pTGl-POLY (som er et miniplasmid på I 10 2;1 kb, der svarer til pTGIH, men indeholder et polylinkersegment fra M13TG131).In replacing G at position -3 with A. The mutated fragment was then inserted between the Eco RI and KpnI sites of the plasmid pTG1-POLY (which is a mini plasmid of I10 2; 1 kb corresponding to pTGIH). but contains a polylinker segment from M13TG131).
KpnI-Hindlll-fragmentet fra P319-13 blev derpå klonet i det samme plasmid (mellem Kpnl og Hindlll), efterfulgt af et Hindlll-Xhol-fragment fra P319-6 (mel- lem Hindlll og Sall), hvilket gav en komplet env-kodende sekvens flankeret af to 15 Pstl-steder (plasmidet pTG1124).The KpnI-HindIII fragment from P319-13 was then cloned into the same plasmid (between KpnI and HindIII), followed by a HindIII-XhoI fragment from P319-6 (between HindIII and SalI) to give a complete env. coding sequence flanked by two 15 Pst I sites (plasmid pTG1124).
Indføring af disse to Pstl-restriktionssteder muliggør en nemmere manipulation af I DNA fra env-genet under den efterfølgende konstruktion. Som anført ovenfor kræver ekspression af et heterologt protein i vacciniavirus, at den kodende sekvens I 20 anbringes sammen med en vacciniapromotorsekvens og indsættes i et ikke- essentielt vaccinia-DNA-segment. Dette DNA anbragt på begge sider muliggør H rekombination med vaccinia-genomet in vivo ved en dobbelt reciprok rekombi- I nation, som overfører den kodende sekvens og den ledsagende promotor til vaccinia-genomet.Introduction of these two PstI restriction sites allows for easier manipulation of the I DNA from the env gene during subsequent construction. As indicated above, expression of a heterologous protein in vaccinia virus requires that the coding sequence I 20 be paired with a vaccinia promoter sequence and inserted into a nonessential vaccinia DNA segment. This DNA, located on both sides, enables H recombination with the vaccinia genome in vivo by a double reciprocal recombination that transfers the coding sequence and the accompanying promoter to the vaccinia genome.
2525
Til dette formål blev ovennævnte Pstl-Pstl-fragment klonet i Pstl-stedet på I pTG186-POLY. Herved fås et plasmid, der betegnes pTG1125.For this purpose, the above PstI-Pstl fragment was cloned into the PstI site of I pTG186-POLY. Thereby a plasmid designated pTG1125 is obtained.
I Plasmidet pTG186-POLY kan dannes ud fra plasmidet pTG188, der spaltes med PstIIn the plasmid pTG186-POLY can be formed from the plasmid pTG188 digested with PstI
I 30 og religeres ved hjælp af T4-ligase.In 30 and relegated by T4 ligase.
Plasmidet pTG188 blev den 20. juni 1985 deponeret hos Collection Nationale de I Cultures de Microorganismes de l'Institut Pasteur, 28, rue du Docteur Roux, 75015 I Paris, Frankrig, med følgende nummer: I 35 DK 175613 B1 15 E. coli 5K pTG188: nr. I 458.The plasmid pTG188 was deposited on June 20, 1985, at the Collection Nationale de I Cultures de Microorganisms de l'Institut Pasteur, 28, rue du Docteur Roux, 75015 in Paris, France, with the following number: I 35 DK 175613 B1 15 E. coli 5K pTG188: No. I 458.
Overførsel af den kodende sekvens for env-genet og den ledsagende . promotor til vaccinia-genomet udføres som følger.Transmission of the coding sequence for the env gene and the companion. promoter for the vaccinia genome is performed as follows.
5 EKSEMPEL 3EXAMPLE 3
Kloning i vacciniavirus til dannelse af W.TG.e LAV 9-1 10Cloning in vaccinia virus to form W.TG.e LAV 9-1 10
Den af Smith et al. (1983) beskrevne strategi bygger pi udskiftning in vivo af vildtype-virusgenomet med et plasmid, der bærer en indsætning i VV-TK-genet, således at det TK-gen, der bæres af viruset, inaktiveres. TK'-vira kan selekteres ved udpladning på en cellelinje (TK-negativ) i nærværelse af 5-bromdeoxyuridin 15 (5BUDR) (Mackett et al., 1982). Thymidinkinase phosphorylerer 5BUDR til 5'-monophosphat, som derefter omdannes til triphosphat. Denne forbindelse er en analog til dTTP, og inkorporering deraf i DNA blokererden korrekte udvikling af viruset. Et TK1/4—virus kan ikke desto mindre replikere sit DNA på normal vis, og dette fører til viruszoner, der er synlige i en ligeledes TK~ cellelinje.The one by Smith et al. (1983) strategy builds on in vivo replacement of the wild-type virus genome with a plasmid carrying an insertion into the VV-TK gene, thereby inactivating the TK gene carried by the virus. TK 'viruses can be selected by plating on a cell line (TK negative) in the presence of 5-bromodeoxyuridine 15 (5BUDR) (Mackett et al., 1982). Thymidine kinase phosphorylates 5BUDR to 5'-monophosphate, which is then converted to triphosphate. This compound is an analogue of dTTP, and its incorporation into the DNA blocks the proper development of the virus. Nevertheless, a TK1 / 4 virus can replicate its DNA in the normal way, and this leads to virus zones that are visible in a similar TK cell line.
2020
Vacciniavirus propageres i cytoplasmaet i de inficerede celler og ikke i deres kerne.Vaccinia viruses are propagated in the cytoplasm of the infected cells and not in their nucleus.
Det er derfor ikke muligt at udnytte værts-DNA'ets replikations- og transkriptions-maskineri, og virionen skal have bestanddelene til ekspression af sit genomet. Oprenset W-DNA er ikke infektiøst.Therefore, it is not possible to utilize the host DNA's replication and transcription machinery, and the virion must have the components to express its genome. Purified W-DNA is not infectious.
2525
For at fremstille rekombinanter er det nødvendigt samtidigt at udføre celleinfektion med VV-virionen og en transfektion med det klonede DNA-segment, som har interesse. Fremstillingen af rekombinanter er imidlertid begrænset til den lille del af cellerne, som er kompetente for transfektion med DNA. Det var derfor nødvendigt at 30 iværksætte en indirekte "kongruens‘’-strategi for at reducere baggrundsstøj fra ikke-rekombinante stamvira. Dette blev udført ved som levende infektiøs virus at anvende en varmefølsom (ts) vacciniamutant, som ikke kan formere sig ved en ikke-permissiv temperatur på 39,5°C (Drillien og Spehner, 1983). Når cellerne inficeres med en ts-mutant under ikke-permissive betingelser og transficeres med 35 DNA fra et vildtype-virus, sker virusmultiplikationen kun i de celler, som er kom- I DK 175613 B1 I 16 I petente for transfektionen, og i hvilke der er forekommet en rekombination I mellem vildtypevirusets DNA og ts-virusgenomet; intet virus vil blive multipliceret i de andre celler pi trods af, at de er blevet inficeret. Hvis et rekombinant plasmid indeholdende et vaccinia-DNA-fragment såsom pTG1125 inkorporeres i 5 transfektionsblandingen i en passende koncentration med vildtype-DNA, er det også muligt at opnå, at det deltager i den homologe rekombination med vaccinia- DNA’et i de kompetente celler.To prepare recombinants, it is necessary to simultaneously carry out cell infection with the VV virion and a transfection with the cloned DNA segment of interest. However, the production of recombinants is limited to the small portion of cells competent for transfection with DNA. Therefore, it was necessary to implement an indirect "congruence" strategy to reduce background noise from non-recombinant progenitor viruses, which was performed by using as a live infectious virus a heat-sensitive (ts) vaccine mutant that cannot reproduce by a non-recombinant -permissive temperature of 39.5 ° C (Drillien and Spehner, 1983) When the cells are infected with a ts mutant under non-permissive conditions and transfected with 35 DNA from a wild-type virus, the virus multiplication occurs only in the cells that are In the case of the transfection, in which there is a recombination I between the wild-type virus DNA and the ts virus genome, no virus will be multiplied in the other cells p despite being infected. a recombinant plasmid containing a vaccinia DNA fragment such as pTG1125 is incorporated into the transfection mixture at an appropriate concentration with wild-type DNA, it is also possible to achieve its participation in the homologous gene. combination with the vaccinia DNA in the competent cells.
Monolag af primære hønsefoster-fibroblastceller (CEF) inficeres ved 33°C med W- 10 København ts7 (0,1 pfu/celle) og transficeres med et calciumphosphat-copræcipitat af DNA fra vildtype-W-København-virus (50 ng/106 celler) og det rekombinante I plasmid (50 ng/106 celler).Monolayers of primary chicken embryo fibroblast cells (CEF) are infected at 33 ° C with W-10 Copenhagen ts7 (0.1 pfu / cell) and transfected with a calcium phosphate coprecipitate of wild-type W-Copenhagen virus DNA (50 ng / 106 cells) and the recombinant I plasmid (50 ng / 106 cells).
I Efter 2 timers inkubation ved en temperatur, som ikke tillader udvikling af ts- I 15 viruset (39,5°C), inkuberes cellerne på ny i 48 timer ved 39,5°C. Fortyndinger af I ts+-virus anvendes til geninfektion af et monolag af humane 143 B-celler ved 37°C, I hvilke celler derefter inkuberes i nærværelse af 5BUDR (150 pg/ml). Forskellige TK‘ -virusplaques fås fra de celler, som har modtaget det rekombinante plasmid, hvor- I imod kontrolkultureme uden plasmid ikke har synlige plaques. TK‘-virus subklones I 20 derefter ved endnu en selektion i nærværelse af 5BUDR. 1 I 35After 2 hours of incubation at a temperature that does not allow the development of the ts-I virus (39.5 ° C), the cells are again incubated for 48 hours at 39.5 ° C. Dilutions of I ts + virus are used to re-infect a monolayer of human 143 B cells at 37 ° C, in which cells are then incubated in the presence of 5BUDR (150 µg / ml). Different TK 'virus plaques are obtained from the cells that have received the recombinant plasmid, whereas the control cultures without plasmid do not have visible plaques. The TK virus is then subcloned into another selection in the presence of 5BUDR. 1 I 35
En korrekt dobbelt reciprok rekombination mellem det hybride plasmid pTG1125 og I W-genomet fører til udskiftning af TK-genet, der bærer indsætningen, med TK- I genet fra viruset, og rekombinanterne bliver således TK\ I 25 I DNA'er oprenset ud fra de forskellige rekombinante TK'-vira spaltes med Hindlll og underkastes elektroforese på agarosegel. DNA-fragmenterne overføres til et I nitroceiluloseRIter ifølge Southern-teknikken (1975). Filtret hybridiseres derpå med I piasmidet pTG1125, der er nick-translateret med 32P. Efter vask af filtret 30 fluorograferes dette, og bånd på 3,85, 2,9 og 0,8 kb er synlige ved autoradiografi, I når vacciniaviruset har inkorporeret, LAV-env-oenet. Én af disse rekombinanter, I W.TG.eLAV 9*1, blev selekteret til følgende undersøgelser.A correct double reciprocal recombination between the hybrid plasmid pTG1125 and the I W genome leads to replacement of the insert-carrying TK gene with the TK-I gene of the virus, thus recombinants are purified from TK \ I 25 I DNAs. the various recombinant TK 'viruses are digested with HindIII and subjected to electrophoresis on agarose gel. The DNA fragments are transferred to a nitroceilulose RIter according to the Southern technique (1975). The filter is then hybridized with the I piasmid pTG1125, which is nick-translated with 32P. After washing the filter 30, this is fluorographed and bands of 3.85, 2.9 and 0.8 kb are visible by autoradiography, when the vaccinia virus has incorporated the LAV env. One of these recombinants, In W.TG.eLAV 9 * 1, was selected for the following studies.
17 DK 175613 B1 EKSEMPEL 4EXAMPLE 4
Env-orotein syntetiseret ud fra et rekombinant vaccinia-LAV-virus 5 For at påvise ekspressionen af LAV-env-aenet ud fra det hybride vacciniavirus inficeres gnaverceller, BHK21, som er dyrket i et G-MEM-medium + 10% føtalt kalveserum, med den omtalte rekombinant, VV.TG.eLAV 9-1.Env protein synthesized from a recombinant vaccinia LAV virus 5 To detect the expression of the LAV env gene from the hybrid vaccinia virus, rodent cells, BHK21, which are grown in a G-MEM medium + 10% fetal calf serum are infected. with the recombinant mentioned, VV.TG.eLAV 9-1.
10 Et frisk semikonfluent monolag (106 celler) inficeres med 0,2 pfu/-celle og inkuberes i 18 timer.A fresh semiconfluent monolayer (106 cells) is infected with 0.2 pfu / cell and incubated for 18 hours.
Mediet fjernes derefter, og der tilsættes et medium med ringe methioninindhold (1 ml pr. 106 celler) beriget med 10 μΙ/ml 35S-methionin. Cellerne inkuberes ved 15 37°C, og de mærkede proteiner opsamles ved centrifugering. Efter opdeling i bundfald og supernatant inkuberes proteinerne med et serum fra en AIDS-patient.The medium is then removed and a medium of low methionine content (1 ml per 106 cells) is added enriched with 10 μΙ / ml of 35 S-methionine. The cells are incubated at 37 ° C and the labeled proteins are collected by centrifugation. After partitioning and supernatant, the proteins are incubated with a serum of an AIDS patient.
De proteiner, der reagerer med serummet, isoleres ved adsorption på en protein A/sepharose-harpiks og udplades ved elektroforese på en SDS-polyacrylamidgel og autoradiograferes i henhold til en teknik, der er beskrevet af Lathe et al., 1980.The proteins that react with the serum are isolated by adsorption on a protein A / sepharose resin and plated by electrophoresis on an SDS-polyacrylamide gel and autoradiographed according to a technique described by Lathe et al., 1980.
20 Autoradiografierne viser, at serummet fra AIDS-patienten specifikt binder tre proteiner fra de inficerede celleekstrakter (resultatet er identisk med eller svarer til det, der fås med andre patientsera). De tilnærmede molekylvægte på 160, 120 og 41 kD antyder ækvivalens med de gpl60-, gpl20- og gp41-bånd, der identificeres af sera fra AIDS-patienter, i en præparation af autentisk gnv-glycoprotein og i cel-25 leekstrakter inficeret med LAV-virus. Denne iagttagelse, at tre proteiner udtrykkes fra den rekombinante vektor, der kun bærer den sekvens, som koder for LAV-env-genet, underbygger hypotesen om, at gpl20 og gp41 dannes ved proteolytisk spaltning af det primære gpl60-translationsprodukt.The autoradiographs show that the serum of the AIDS patient specifically binds three proteins from the infected cell extracts (the result is identical to or similar to that obtained with other patient sera). The approximate molecular weights of 160, 120 and 41 kD suggest equivalence to the gp160, gp120 and gp41 bands identified by sera from AIDS patients in a preparation of authentic gnv glycoprotein and in cell extracts infected with LAV. -virus. This observation that three proteins are expressed from the recombinant vector carrying only the sequence encoding the LAV env gene supports the hypothesis that gp120 and gp41 are formed by proteolytic cleavage of the primary gp160 translation product.
30 Den sekvens, som koder for env. fører til et primært translationsprodukt på ca.The sequence encoding env. leads to a primary translation product of approx.
90 kD, mens den ved ovenstående fremgangsmåde vundne env-precursor har en tilnærmet molekylvægt på ca. 160 kD. Denne forskel kan tilskrives en meget betydelig glycosylering. Ved spaltning med endoglycosidase F, som eliminerer glycosylgrupperne, har det kunnet påvises, at der var en god korrelation mellem de90 kD, while the env precursor obtained by the above method has an approximate molecular weight of approx. 160 kD. This difference can be attributed to a very significant glycosylation. By cleavage with endoglycosidase F, which eliminates the glycosyl groups, it has been shown that there was a good correlation between the
I DK 175613 B1 II DK 175613 B1 I
I 18 II 18 I
I produkter, der er vundet ifølge den foreliggende opfindelse, og de forventede IIn products won according to the present invention and the expected I
I produkter (fig. 1). IIn products (Fig. 1). IN
I 5 EKSEMPEL 5 IEXAMPLE 5 I
I Påvisning af anti-env-antistoffer hos mus vaccineret med viruset II Detection of anti-env antibodies in mice vaccinated with the virus I
I VV.TC.eLAV 9-1 ' II VV.TC.eLAV 9-1 'I
I 10 5 uger gamle Balb/c-hanmus vaccineres ved subkutan injektion med 5xl07 pfu IIn 10 5 week old male Balb / c mice are vaccinated by subcutaneous injection with 5x107 pfu I
I VV.TG.eLAV 9-1-virus pr. dyr. De modtager endnu en injektion med samme dosis IIn VV.TG.eLAV 9-1 virus per animals. They receive another injection at the same dose I
I efter 2 uger, og der tages blod 1, 2 og 4 uger efter den anden injektion. Der søges IAfter 2 weeks and blood is taken 1, 2 and 4 weeks after the second injection. You are looking for
I efter tilstedeværelsen af anti-stoffer rettet mod LAV-virus- og vacciniavirusdeter- IIn the presence of antibodies directed against LAV virus and vaccinia virus detergent I
I minanter i deres serum. IIn minants in their serum. IN
I 15 II 15 I
I Alle de vaccinerede dyr giver sera, der kan reagere med vacciniavirus i en ELISA- IIn All vaccinated animals give sera that can react with vaccinia virus in an ELISA-I
I test. Derimod er responset i ELlSA-testen mod LAV-virus svagt og kun reproducer- IIn test. In contrast, the response in the EL1SA test against LAV virus is weak and only reproducible
I bart i ringe grad. For at forbedre testenes følsomhed blev der anvendt en "western ITo a lesser extent. To improve the sensitivity of the tests, a "western I" was used
I blot"-teknik. Ved denne metode kan der påvises antistoffer, der er i stand til at IIn this technique, antibodies capable of detecting antibodies can be detected
I 20 reagere med LAV-virusproteinerne, efter at disse er blevet denatureret med SDS i II react with the LAV virus proteins after being denatured with SDS in I
I en elektroforesegel og overført til en nitrocellulosemembran. I dette forsøg er de IIn an electrophoresis seal and transferred to a nitrocellulose membrane. In this experiment they are
I anvendte nitrocellulosemembraner fra et LAV-BLOT-kit, der forhandles af II used nitrocellulose membranes from a LAV-BLOT kit sold by I
I Diagnostic Pasteur, og til hvilke LAV-virusproteinerne allerede er fæstnet. Disse IIn the Diagnostic Pasteur and to which the LAV virus proteins are already attached. These I
I membraner skæres op i bånd, og hvert bånd inkuberes med serum fra de vaccine- IIn membranes, cut into bands and each band incubated with serum from the vaccine I
I 25 rede mus (1/20 fortynding). Et andet antistof (fåre-antimus) bundet til peroxidase IIn 25 prepared mice (1/20 dilution). Another antibody (sheep antimus) bound to peroxidase I
I gør det muligt at visualisere de LAV-virusproteiner, som har bundet rhuseanti- IYou make it possible to visualize the LAV virus proteins that have bound rhus antigen
stoffer. Isubstances. IN
I Flere sera (12/27) giver en specifik reaktion med et protein med en molekylvægt IIn Several sera (12/27), a specific reaction with a protein with a molecular weight I gives
I 30 på ca. 160 kD svarende til gpl60 fra env (fig. 2). I et vist antal sera ses også en IIn 30 of approx. 160 kD corresponding to gp160 from env (Fig. 2). In a certain number of sera an I is also seen
I reaktion med proteinet gp41. Det skal bemærkes, at seraene fra nogle mus ved IIn reaction with the protein gp41. It should be noted that the sera of some mice are known to you
I Western blot giver signaler svarende til uidentificerede proteiner i LAV-viruspræpa- IIn Western blot, signals corresponding to unidentified proteins in LAV virus prepress I
I rationen fæstnet til membranerne. IIn the ration attached to the membranes. IN
I 35 II 35 I
19 DK 175613 B1 EKSEMPEL 6EXAMPLE 6
Konstruktion af PTG1128 5 Dette plasmid pTG1128 er identisk med plasmidet pTG1125 bortset fra, at den sekvens, der koder for transmembranzonen, er blevet muteret for at erstatte arginin med isoleucin, hvilket er sket for at forbedre proteinets fæstnelse til cellemembranen.Construction of PTG1128 This plasmid pTG1128 is identical to plasmid pTG1125 except that the sequence encoding the transmembrane zone has been mutated to replace arginine with isoleucine, which has been done to enhance the attachment of the protein to the cell membrane.
10 Hindlll-BamHI-fragmentet fra pTG1124 indeholdende den i eksempel 2 beskrevne env-transmembranzone indsættes i fagen M13TG131 efter spaltning med Hindlll-BamHI. Dette giver fagen Ml3TG154.The HindIII-BamHI fragment from pTG1124 containing the env transmembrane zone described in Example 2 is inserted into the phage M13TG131 after cleavage with HindIII-BamHI. This gives the phage Ml3TG154.
På denne fag M13TG154 udføres derefter en lokaliseret mutagenese, der skal er-15 statte den kodon, der koder for arginin, med en kodon, der koder for isoleucin. Til dette formål anvendes følgende oligonukleotid: 5' GGTTTAATAATAGTTTT 3’.In this subject M13TG154, a localized mutagenesis is then performed to replace the arginine-encoding codon with an isoleucine-encoded codon. For this purpose, the following oligonucleotide is used: 5 'GGTTTAATAATAGTTTT 3'.
20 Dette giver fagen M13TG155, idet sekvenserne er blevet muteret som følger:This yields the phage M13TG155, the sequences having been mutated as follows:
Gly Leu Arg Ile ValGly Leu Arg Ile Val
Oprindelig sekvens: GGT TTA AGA ATA GTTOriginal sequence: GGT TTA AGA ATA GTT
Muteret sekvens: GGT TTA ATA ATA GTTMutated sequence: GGT TTA ATA ATA GTT
25 Ile25 Ile
Det således muterede BamHI-Hindlll-fragment overføres fra M13TG155 til plasmidet pTG1124 i de tilsvarende steder, hvilket giver plasmidet pTG1127, som rekonstituerer gny-genet som tidligere med undtagelse af, at kodonen for arginin 30 er erstattet med en isoleucin-kodon.The thus mutated Bam HI-HindIII fragment is transferred from M13TG155 to the plasmid pTG1124 at the corresponding sites, giving the plasmid pTG1127, which reconstitutes the gny gene as before except that the codon for arginine 30 is replaced by an isoleucine codon.
Som beskrevet i eksempel 1 klones Pstl-Pstl-fragmentet fra pTG1127 i Pstl-stedet på plasmidet pTG186-POLY, hvilket giver plasmidet pTG1128.As described in Example 1, the Pstl-Pstl fragment from pTG1127 is cloned into the Pstl site of plasmid pTG186-POLY to yield plasmid pTG1128.
DK 175613 B1 IDK 175613 B1 I
I 20 II 20 I
I EKSEMPEL 7 IEXAMPLE 7 I
I Konstruktion af plasmidet PTG1130 IConstruction of the plasmid PTG1130 I
I 5 I dette plasmid fusioneres den sekvens, som koder for rabiesglycoproteinets IIn this plasmid, the sequence encoding the rabies glycoprotein I is fused
I transmembranzone, med begyndelsen af den sekvens, der koder for den hydrofobe IIn the transmembrane zone, with the beginning of the sequence encoding the hydrophobic I
I del af env-glycoproteinet. IIn part of the env glycoprotein. IN
I Rabiesglycoproteinets transmembranzone stammer fra et BamHI-Pstl-fragment på IThe transmembrane zone of the Rabies glycoprotein is derived from a BamHI-Pst I fragment of I
I 10 fagen M13TGRG151. IIn the phage M13TGRG151. IN
I Dette fragment klones i fagen M13TG154 mellem BamHI- og Pstl-stederne (se IThis fragment is cloned into phage M13TG154 between the Bam HI and Pst I sites (see I
I forrige eksempel). Dette giver fagen M13TG156. IIn the previous example). This gives the subject M13TG156. IN
I 15 Derefter udføres en lokaliseret mutagenese på M13TG156 for i fase at fusionere IThen, a localized mutagenesis on M13TG156 is performed to merge in phase I
I env- og rabiessekvenserne med et oligonukleotid, idet der dannes en sløjfe IIn the env and rabies sequences with an oligonucleotide, forming a loop I
I 5' GCTGTGGTATATAAAATATGTATTACTGAGTG 3’ II 5 'GCTGTGGTATATAAAATATGTATTACTGAGTG 3' I
I Tyr Leu Lys Ile Phe Gly Lys Tyr Val II Tyr Leu Lys Ile Phe Gly Lys Tyr Val I
I 20 TAT ATA AAA ATA TTC------GGG AAG TAT GTA II 20 TAT ATA AAA ATA TTC ------ GGG AAG TAT GTA I
I x. X---I x. X ---
I tm env *· -r tm rabies II tm env * · -r tm rabies I
I Dette giver således fagen M13TG157. IThus, this gives the subject M13TG157. IN
I 25 II 25 I
I Rabiesglycoproteinets transmembranzone (tm), som lige er blevet fusioneret med IIn the Rabies glycoprotein transmembrane zone (tm), which has just been merged with I
I eny-genet, overføres derefter til plasmidet pTGl 124. IIn the eny gene, it is then transferred to the plasmid pTG1 124
I Til dette formål klones Hindlll-BgIII-fragmentet fra M13TG157 i pTGil24, hvori IFor this purpose, the HindIII-BgIII fragment of M13TG157 is cloned into pTGil24, wherein I
I 30 der er foretaget en Hindlll-BamHI-spaltning (dette nedbryder BamHI- og Bglll- IIn Hindlll-BamHI cleavage (this breaks down BamHI and BglII-I
I stederne). IIn the places). IN
I Blgll-stedet på M13TG157 stammer fra rabies-gp-fragmentet: IThe BlgII site of M13TG157 is derived from the rabies gp fragment: I
I 35 II 35 I
I DK 175613 B1 I 21 I BamHI Bglu I I-V77//S//Λ-—^ I t.n. ?stl I 5 I Pa denne måde fås plasmidet pTGl 126.I DK 175613 B1 I 21 I BamHI Bglu I I-V77 // S // Λ -— ^ I t.n. In this way, plasmid pTGl126 is obtained.
I Som tidligere klones Pstl-Pstl-fragmentet fra pTG1126 i Pstl-stedet på pTG186- I POLY til dannelse af plasmidet pTG1130.As before, the PstI-Pstl fragment from pTG1126 is cloned into the Pstl site of pTG186-I POLY to generate the plasmid pTG1130.
I 10 I EKSEMPEL 8 I Konstruktion af PTG1131 I 15I 10 I EXAMPLE 8 I Construction of PTG1131 I 15
I Formålet med konstruktionen af dette plasmid er at fusionere signalsekvensen fra IThe purpose of constructing this plasmid is to merge the signal sequence from I
I env-genet og signalsekvensen fra rabiesglycoproteinet. IIn the env gene and signal sequence from the rabies glycoprotein. IN
I Signalsekvensen fra rabiesglycoproteinet fjernes fra plasmidet pTG155 PRO i form IIn the Rabies glycoprotein signal sequence, the plasmid pTG155 PRO in Form I is removed
I 20 af et BgHI-HindllTfragment, som klones i Pstl-HindW-stederne på M13TG130 ved IIn 20 of a BgHI-HindIII fragment cloned into the PstI-HindW sites of M13TG130 by I
I hjælp af en enkeltstrenget adaptor med følgende sekvens: IUsing a single-stranded adapter with the following sequence:
I 5' GATCTGCA 3’ I Dette giver fagen M13TG158.I 5 'GATCTGCA 3' I This gives the phage M13TG158.
I 25 I Overførsel af eny-signalpeptidet til M13TG158 udføres derefter for at fusionere sidstnævnte med det gen, der koder for signalpeptidet fra rabiesglycoproteinet.I 25 I Transfer of the eny signal peptide to M13TG158 is then performed to fuse the latter with the gene encoding the signal peptide from the rabies glycoprotein.
Til dette formål udføres kloning af PstI-fragmentet, der er behandlet med SI-30 nuklease og derefter med Klenow og Kpnl, i M13TG158 spaltet med Hindlll behandlet med Klenow-Kpnl: 35For this purpose, cloning of the PstI fragment treated with SI-30 nuclease and then with Klenow and KpnI is performed in M13TG158 digested with HindIII treated with Klenow-KpnI: 35
DK 175613 B1 IDK 175613 B1 I
22 I22 I
EcoRI Kpnl Pst*/HindIII° Pst*/BglII* IEcoRI Kpnl Pst * / HindIII ° Pst * / BglII * I
M13 TGi30 —|-1—-EZZZD—^-\n//rihi- IM13 TGi30 - | -1 —- EZZZD - ^ - \ n // rihi- I
signal signal Isignal signal I
env rabies Ienv rabies I
Dette giver plasmidet M13TG159. IThis yields the plasmid M13TG159. IN
10 I10 I
I KpnI-Pstl-blokken fra M13TG159 overføres til M13TG131 til dannelse af Plasmidet IIn the KpnI-Pstl block of M13TG159, transfer to M13TG131 to form the plasmid I
M13TG160. IM13TG160. IN
I Pst ^Bgl11 Hinde/Pst* K - EcoRIIn Pst ^ Bgl11 Hind / Pst * K - EcoRI
I M13 TGI 31-ZZZZZZZZ^—*-ViTTTt/X-(-1—In M13 TGI 31-ZZZZZZZZ ^ - * - ViTTTt / X - (- 1-
I signal signal IIn signal signal I
I rabies env I 20 > -—> I En lokaliseret mutagenese på M13TG160 gør det muligt i fase at fusionere env- I sekvenserne og rabiesglycoproteinet (idet der dannes en sløjfe). Dette skyldes oligonukfeotidet I 25 5' GACCCACAATTTTTCTGTAATAGGGAATTTCCCAAA 3 ’ I Signal rabies envIn rabies env I 20> -—> I A localized mutagenesis on the M13TG160 allows in phase fusion of the env- I sequences and the rabies glycoprotein (forming a loop). This is due to the oligonucleotide I 25 5 'GACCCACAATTTTTCTGTAATAGGGAATTTCCCAAA 3' I Signal rabies env
Cys Phe { Gly Lys Phe Pro Ile Tyr Thr Gin Lys Leu M 5' TGT TTT GGG AAA TTC CCT ATT TAC---ACA__G AA AAA TTG 3 x I 35 Dette giver fagen M13TG161.Cys Phe {Gly Lys Phe Pro Ile Tyr Thr Gin Lys Leu M 5 'TGT TTT GGG AAA TTC CCT ATT TAC --- ACA__G AA AAA TTG 3 x I 35 This gives the phage M13TG161.
23 DK 175613 B123 DK 175613 B1
PvuII-Kpnl-fragmentet fra M13TG161 klones derefter i pTGH26 spaltet med EcoRI behandlet med Klenow-Kpnl (PvuII-stedet i M13TG161 stammer Fra M13 i den region, der findes opstrøms for polylinkeren). Dette giver plasmidet pTGH29.The PvuII-Kpnl fragment from M13TG161 is then cloned into pTGH26 digested with EcoRI treated with Klenow-Kpnl (the PvuII site of M13TG161 originates from M13 in the region located upstream of the polylinker). This yields the plasmid pTGH29.
55
Ved kloning af Pstl-Pstl-fragmentet fra pTG1129 i plasmidet pTGl86-POLY spaltet med PstI fås plasmidet pTG1131.By cloning the PstI-Pstl fragment from pTG1129 into plasmid pTGl86-POLY digested with PstI, plasmid pTG1131 is obtained.
10 EKSEMPEL 9EXAMPLE 9
Fremstilling af plasmidet PTG1132Preparation of the plasmid PTG1132
Ved kloning af Pstl-Pstl-fragmentet fra pTG1128 i Pstl-stedet på M13TG131 fås 15 plasmidet M13TG162.By cloning the Pstl-Pstl fragment from pTG1128 at the Pstl site of M13TG131, plasmid M13TG162 is obtained.
Der foretages derefter en lokaliseret mutagenese ved hjælp af oligo-nukleotidet 5’ ATT CCCACT GCTTAGTATTC ATT CTGCACCACT C 3' 20A localized mutagenesis is then carried out by the oligonucleotide 5 'ATT CCCACT GCTTAGTATTC ATT CTGCACCACT C 3' 20
Dette gør det muligt at anbringe en stopkodon i enden af gpl20. De vundne sekvenser er som følger:This makes it possible to place a stop codon at the end of gp120. The sequences won are as follows:
R E K R IR E K R I
Oprindelig sekvens s CAG ^VGA GAA ^ AAA GCA GTGOriginal sequence s CAG ^ VGA GAA ^ AAA GCA GTG
Formodet spaltningssted i gpl20 N E Y xxxPresumed cleavage site in gpl20 N E Y xxx
Muteret sekvens : CAG AAT GAA TAC TAA GCA GTGMutated sequence: CAG AAT GAA TAC TAA GCA GTG
Dette giver fagen M13TG168.This gives the subject M13TG168.
I DK 175613 B1 II DK 175613 B1 I
I 24 II 24 I
I Ved genkloning af PstI-fragmentet fra M13TG168 i Pstl-stedet pi pTG186-POLY fås IBy re-cloning the PstI fragment from M13TG168 into the PstI site pi pTG186-POLY, I
I plasmidet pTG1132. IIn the plasmid pTG1132. IN
I 5 EKSEMPEL 10 IEXAMPLE 10 I
I Konstruktion af plasmidet PTG1133 IConstruction of the plasmid PTG1133 I
I Ved lokaliseret mutagenese på M13TG162 ved hjælp af følgende oligonukleotid II By localized mutagenesis on M13TG162 using the following oligonucleotide I
I 10 5' ATTCCCACTGCTTG GTGTTCATTGTGC ACCACTC 3’ II 5 'ATTCCCACTGCTTG GTGTTCATTGTGC ACCACTC 3' I
I fås en bakteriofag, hvor et potentielt spaltningssted, der skiller gpl20 og gp40, er I blevet nedbrudt.You get a bacteriophage where a potential cleavage site that separates gp120 and gp40 has been broken down.
I De modificerede sekvenser er som følger: IThe modified sequences are as follows:
I 15 II 15 I
I Oprindelig sekvens . CAG AGA G AA AAA AGA * GCA GTG IIn Original Sequence. CAG AGA G AA AAA AGA * GCA GTG I
I rekrTav IIn record I
I spaltningssted IIn cleavage site I
I ' 20 II '20 I
I Muteret sekvens - CAG AAT GAA CAC CAA GCA IIn Mutant Sequence - CAG AAT GAA CAC CAA GCA I
I N E H Q II N E H Q I
I 25 Dette giver fagen M13TG165. II 25 This gives the subject M13TG165. IN
I Ved genkloning af Pstl-Pstl-fragmentet fra M13TG165 i pTG186-POLY i Pstl-stedet II By re-cloning the Pstl-Pstl fragment from M13TG165 into pTG186-POLY at the Pstl site I
I fås plasmidet pTGl 133. IThe plasmid pTGl 133. is obtained
I 30 II 30 I
I 35 II 35 I
EKSEMPEL 11 25EXAMPLE 11 25
Konstruktion af plasmidet pTGll34 DK 175613 B1 5 Ved kloning af Pstl-Pstl-fragmentet fra pTG1131 i Pstl-stedet på M13TG131 fås fagen M13TG163.Construction of the plasmid pTG1134 DK 175613 B1 5 By cloning the Pstl-Pstl fragment from pTG1131 at the Pstl site of M13TG131, the phage M13TG163 is obtained.
Der udføres en lokaliseret mutagenese på M13TG163 for at nedbryde det samme spaltningssted i gpl20 som ovenfor. Til dette formål anvendes oligonukleotidet 10 5' ATTCCCACTGCTTGATGTTCATTCTGCACCACTC 3'A localized mutagenesis on M13TG163 is performed to break down the same cleavage site in gp120 as above. For this purpose, the oligonucleotide 10 5 'ATTCCCACTGCTTGATGTTCATTCTGCACCACTC 3' is used.
Dette gør det muligt at modificere sekvenserne på følgende måde:This allows the sequences to be modified as follows:
R E K RR E K R
15 Oprindelig sekvens: CAG AGA GAA AAA AGA^GCA GTG15 Original sequence: CAG AGA GAA AAA AGA ^ GCA GTG
Muteret sekvens: CAG AAT GAA CAT CAA GCAMutated sequence: CAG AAT GAA CAT CAA GCA
N E H QN E H Q
20 Under disse betingelser fås fagen M13TG166.Under these conditions, the subject M13TG166 is obtained.
Ved genkloning af Pstl-Pstl-fragmentet i denne fag M13TG166 i Pstl-stedet på pTG186-POLY fås plasmidet pTGl 134.By re-cloning the Pstl-Pstl fragment in this phage M13TG166 at the Pstl site of pTG186-POLY, plasmid pTGl 134 is obtained.
25 EKSEMPEL 12EXAMPLE 12
Immunudfældnina af proteiner syntetiseret af rekombinante VV.TG. eLAV-vira 30Immunoprecipitation of proteins synthesized by recombinant VV.TG. eLAV viruses 30
Ved at gå frem som beskrevet ovenfor for plasmidet pTG1125 fås de hybride vacciniavektorer svarende til de forskellige plasmider, der er fremstillet ovenfor.Proceeding as described above for plasmid pTG1125, the hybrid vaccinia vectors corresponding to the different plasmids prepared above are obtained.
Disse virusvektorer har følgende betegnelser: 35These virus vectors have the following designations:
I DK 175613 B1 II DK 175613 B1 I
I 26 1I 26 1
I W.TG. eLAV 1128 IIn W.TG. eLAV 1128 I
I W.TG. eLAV 1130 IIn W.TG. eLAV 1130 I
I W.TG. eLAV 1131 IIn W.TG. eLAV 1131 I
I W.TG. eLAV 1132 IIn W.TG. eLAV 1132 I
I 5 W.TG. eLAV 1133 IIn 5 W.TG. eLAV 1133 I
I W.TG. eLAV 1134 IIn W.TG. eLAV 1134 I
I De som ovenfor beskrevet vundne proteiner testes ved immunudfældning (fig. 3). IThe proteins obtained as described above are tested by immunoprecipitation (Fig. 3). IN
I 10 Ved alle immunpræcipitaterne fremkommer der for viruset 9-1 en immunudfæld- IIn all the immunoprecipitates, the virus 9-1 produces an immunoprecipitate
I ning svarende til gpl60, gpl20 og gp41. IIn accordance with gpl60, gpl20 and gp41. IN
I Det samme gælder for virus 1128. IThe same applies to viruses 1128. I
I 15 Virus 1130 viser også et gpl60 og et gpl20. IVirus 1530 also shows a gp160 and a gp120. IN
I Proteinet svarende til gp41 har en smule lavere vægt på grund af modifikationen af IThe protein corresponding to gp41 has slightly lower weight due to the modification of I
I dets C-terminale ende. IAt its C-terminal end. IN
I 20 Virus 1131 har et spektrum, der er overvejende identisk med det, der fås for virus II Virus 1131 has a spectrum that is essentially identical to that available for Virus I
I 1130. II 1130. I
I Virus 1132 har naturligvis intet protein svarende til gp41. Proteinet på 105 kD, der IOf course, Virus 1132 has no protein similar to gp41. The 105 kD protein that I
I er til stede i bundfaldet, er en isoform af gpl20 (anderledes glycosylering). IYou are present in the precipitate is an isoform of gp120 (different glycosylation). IN
I 25 II 25 I
I Hvad angår virus 1133, har dette ganske vist proteinerne 160, 120 og 41, men IIn the case of virus 1133, this has proteins 160, 120 and 41, but I
I båndene svarende til proteinerne 120 og 41 er lidt svagere end i de andre spektra. IIn the bands corresponding to proteins 120 and 41 are slightly weaker than in the other spectra. IN
I Det samme gælder virus 1134, hvor gp41 også har en lavere molekylvægt, men IThe same applies to virus 1134, where gp41 also has a lower molecular weight but I
I 30 for hvilket det er klart, at spaltningen er sket med en langsommere kinetik end for II 30 for which it is clear that the cleavage occurred with a slower kinetics than for I
I viraene W.TG. 1125 (9-1) til 1131. IIn the viruses W.TG. 1125 (9-1) to 1131. I
I 35 II 35 I
Konstruktion af plasmidet dT61135 27 DK 175613 B1 EKSEMPEL 13 5 De udskillelseskinetikker, der udføres på viraene VV.TG.eLAV1133 og 1134, viser, at selv om kinetikken for spaltning mellem gpl20 og gp40 er langsommere, finder spaltningen dog sted. Undersøgelse af DNA-sékvensen fra env-aenet viser et andet potentielt spaltningssted (KRR) 8 aminosyrer nedstrøms for det første spaltningssted. Det ser derfor ud til at have betydning, at man muterer dette andet sted for 10 at opnå et rekombinant vacciniavirus, som kun udtrykker gpl60.Construction of the plasmid dT61135 27 DK 175613 B1 EXAMPLE 13 5 The secretion kinetics performed on viruses VV.TG.eLAV1133 and 1134 show that although the kinetics of cleavage between gpl20 and gp40 are slower, however, cleavage occurs. Examination of the DNA sequence from the env gene shows a second potential cleavage site (KRR) 8 amino acids downstream of the first cleavage site. Therefore, it seems important to mutate this second site to obtain a recombinant vaccinia virus expressing only gp160.
Ved lokaliseret mutagenese på M13TG166 ved hjælp af følgende oligo-nukleotid 5’ ATTCTGCACCACGTGATTCTGTGCCTTGGTGGGT 3' fås en fag, i hvilken det andet spaltningssted er modificeret. De modificerede 15 sekvenser er som følger:By localized mutagenesis on M13TG166 by the following oligonucleotide 5 'ATTCTGCACCACGTGATTCTGTGCCTTGGTGGGT 3' a phage is obtained in which the second cleavage site is modified. The modified 15 sequences are as follows:
Oprindelig sekvens: GCA AAG AGA AGA GTGOriginal sequence: GCA AAG AGA AGA GTG
A K R R VA K R R V
t potentielc spaltningsstedt potential cleavage site
Muteret sekvens: GCA CAG AAT CAC · GTGMutated sequence: GCA CAG AAT CAC · GTG
A Q N Η VA Q N Η V
Pstl-Pstl-fragmentet i den vundne fag (M13TG181) klones i pTG186-POLY i Pstl-stedet til dannelse af plasmidet pTG1135.The PstI-Pstl fragment of the obtained phage (M13TG181) is cloned into pTG186-POLY at the Pstl site to generate the plasmid pTG1135.
25 EKSEMPEL 14EXAMPLE 14
Konstruktion af plasmidet PTG1139 30 Den C-terminale del af env-oenet syntetiseret ved hjælp af den rekombinante vacciniavektor W.TG.eLAV1135 er en sekvens, der stammer fra rabiesglycoprote-inet. Det kan derfor være nyttigt også at råde over en anden rekombinant, hvor denne C-terminale del er erstattet med den C-terminale del af eny-genet fra LAV-virus.Construction of plasmid PTG1139 The C-terminal portion of the env synthesized by the recombinant vaccinia vector W.TG.eLAV1135 is a sequence derived from the rabies glycoprotein. Therefore, it may also be useful to have another recombinant in which this C-terminal portion is replaced with the C-terminal portion of the LAV virus eny gene.
3535
I DK 175613 B1 II DK 175613 B1 I
I 28 II 28 I
I Til dette formål udføres der på fagen M13TG165 den samme mutagenese som den, IFor this purpose, the same mutagenesis as I, is carried out on the subject M13TG165
I der blev udført (se eksempel 13) på fagen M13TG166, hvilket giver fagen II was performed (see Example 13) on the phage M13TG166 to give the phage I
I M13TG184. IIn M13TG184. IN
I 5 Pstl-Pstl-fragmentet fra M13TG184 genklones dernæst i plasmidet pTG186-POLY til IIn the 5 Pstl-Pstl fragment of M13TG184, the clone is then cloned into plasmid pTG186-POLY to I
I dannelse af plasmidet pTGl 139.In formation of the plasmid pTG1 139.
I EKSEMPEL 15 IIn Example 15 I
I 10 Konstruktion af plasmidet pTG1136 IConstruction of the plasmid pTG1136 I
I Man vil også gerne have et rekombinant vacciniavirus, der alene udtrykker gpl20. IYou also want a recombinant vaccinia virus that expresses gp120 alone. IN
I Dette gpl20 skulle i modsætning til det, der blev opnået med viruset IIn This gp120 was in contrast to that obtained with virus I
I W.TG.eLAV1132, være forsynet med et C-terminalt forankringsområde. IIn W.TG.eLAV1132, be provided with a C-terminal anchoring area. IN
I 15 II 15 I
I Til dette formål elimineres ved lokaliseret mutagenese de sekvenser, der svarer til II For this purpose, by localized mutagenesis, the sequences corresponding to I are eliminated
I gp40, i fagen M13TG181 ved hjælp af følgende oligonukleotid: IIn gp40, in the phage M13TG181 by the following oligonucleotide: I
I 5' TGCACTCAGTAATACATACACGTGATTCTGTGCCTT 3' II 5 'TGCACTCAGTAATACATACACGTGATTCTGTGCCTT 3' I
I 20 Dette oligonukleotid gør det muligt i fase at fusionere gpl20-sekvenserne (med de II This oligonucleotide allows for phase fusion of the gp120 sequences (with the I
I 2 modificerede spaltningssteder) og sekvenserne af rabiesglycoproteinets trans- IIn 2 modified cleavage sites) and the sequences of the rabies glycoprotein trans I
I membranzone IIn membrane zone I
I KAQ6IHVV F Y VLL II KAQ6IHVV F Y VLL I
I AAG GCA CAG AAT CAC GTG GTG TTCITAT GTA TTA CTG II AAG GCA CAG AAT CAC GTG GTG TTCITAT GTA TTA CTG I
I gpl20 TM rabies II gpl20 TM rabies I
I 30 II 30 I
I Dette giver fagen M13TG182. Pstl-Pstl-fragmentet fra M13TG182 indsættes der- IIn This, the subject gives M13TG182. The PstI-Pstl fragment from M13TG182 is then inserted
I næst i Pstl-stedet i pTGl86-POLY til dannelse af plasmidet pTGl 136. INext in the PstI site of pTGl86-POLY to generate the plasmid pTGl 136.
I 35 II 35 I
Konstruktion af plasmidet PTG1137 EKSEMPEL 16 29 DK 175613 B1 5 Der vides kun lidt om rollen hos den hydrofobe zone, der findes ved den N-terminale del af gp40. Denne zone i env-proteinet kunne være ansvarlig for evnen til induktion af dannelse af syncytia.Construction of the plasmid PTG1137 EXAMPLE 16 29 DK 175613 B1 5 Little is known about the role of the hydrophobic zone found at the N-terminal portion of gp40. This zone of the env protein could be responsible for the ability to induce syncytia formation.
Det synes derfor interessant at fremstille et gpl60, som ikke indeholder denne 10 sekvens.It therefore seems interesting to produce a gp160 which does not contain this sequence.
Til dette formål fusioneres i fase sekvenserne opstrøms og nedstrøms for den del, der koder for dette hydrofobe peptid, i fagen M13TG181 ved hjælp af følgende oligonukleotid: 15 5’ CAATAATTGTCTGGCCTGCACGTGATTCTGTGCCTT 3’To this end, in the phase sequences upstream and downstream of the portion encoding this hydrophobic peptide, the phage M13TG181 is fused by the following oligonucleotide: 15 'CAATAATTGTCTGGCCTGCACGTGATTCTGTGCCTT 3'
Dette gør det muligt at opnå fagen M13TG183, idet følgende fusion udføres:This allows the phage M13TG183 to be obtained by performing the following fusion:
20 AAG GCA CAG AAT CAC GTG GTG — GTA CAG GCC AGA CAA20 AAG GCA CAG AAT CAC GTG GTG - GTA CAG GCC AGA CAA
KAQ NHVV VQARQKAQ NHVV VQARQ
gpl20 hydrofil del 25 ' af gp40gp120 hydrophilic portion 25 'of gp40
Pstl-Pstl-fragmentet fra M13TG183 genklones i Pstl-stedet på pTG186-POLY til dannelse af plasmidet pTG1137.The Pstl-Pstl fragment from M13TG183 is re-cloned into the Pstl site of pTG186-POLY to generate the plasmid pTG1137.
30 3530 35
I DK 175613 B1 II DK 175613 B1 I
I 30 II 30 I
I EKSEMPEL 17 IEXAMPLE 17 I
IIN
I Konstruktion af plasmidet pTG1138 IConstruction of the plasmid pTG1138 I
I 5 Ud over rekombinante vira, der udtrykker gpl60 eller gpl20, kan det være nyttigt IIn addition to recombinant viruses expressing gp160 or gp120, it may be useful
I at fremstille et rekombinant vacciniavirus, der kun udtrykker gp40. IIn producing a recombinant vaccinia virus expressing only gp40. IN
I Til dette formål fusioneres de sekvenser, der koder for signalpeptidet, med de ko- IFor this purpose, the sequences encoding the signal peptide are merged with the codons
I dende sekvenser af gp40 på fagen M13TG163 ved hjælp af følgende oligonukleotid: IIn the same sequences of gp40 on the phage M13TG163 using the following oligonucleotide: I
I 10 II 10 I
I 5’ CAATAATTGTCTGGCCT G AATAG GG AATTT CC C A A A 3’ II 5 'CAATAATTGTCTGGCCT G AATAG GG AATTT CC C A A A 3' I
I Dette gør det muligt at fremstille fagen M13TG180, som omfatter følgende fusion: IThis makes it possible to prepare the phage M13TG180, which comprises the following fusion:
I 15 TGT TTT GGG AAA TTC -f— C AG GCC AG A C AA II 15 TGT TTT GGG AAA TTC -f— C AG GCC AG A C AA I
I 'CF.GKP.QARQ' IIn 'CF.GKP.QARQ' I
I signal gp40 (hydrofil del) IIn signal gp40 (hydrophilic part) I
I 20 fra II 20 from I
rabies-gp Irabies-gp I
I Pstl-Pstl-fragmentet fra M13TG180 indsættes i Pstl-stedet på pTG186-POLY til IIn the Pstl-Pstl fragment of M13TG180, the Pstl site of pTG186-POLY is inserted into I
I dannelse af plasmidet pTGl 138. IIn formation of the plasmid pTG1 138. I
I 25 II 25 I
I EKSEMPEL 18 IIn Example 18 I
I Konstruktion af plasmidet PTG1162 IConstruction of the plasmid PTG1162 I
I 30 Som i tilfælde af gpl60 (plasmidet pTGl 139) kan det også være vigtigt at råde IAs in the case of gp160 (plasmid pTG1139), it may also be important to advise I
I over et rekombinant virus, der udtrykker et gp40, i hvilket forankringszonen og IIn over a recombinant virus expressing a gp40 in which the anchoring zone and I
I den intracytoplasmiske zone er sekvenser af env-oenet fra LAV-virus og ikke de IIn the intracytoplasmic zone, sequences of the env from the LAV virus and not the
I tilsvarende sekvenser fra rabiesglyco-proteinet. IIn similar sequences from the rabies glyco protein. IN
DK 175613 B1 31DK 175613 B1 31
For at opnå dette erstattes Hindlll-BgII-fragmentet i M13TG180 med Hindlll-Bgll-fragmentet fra M13TG165, hvilket giver fagen M13TG190.To achieve this, the HindIII-BgII fragment of M13TG180 is replaced by the HindIII-BgII fragment of M13TG165 to give the phage M13TG190.
Plasmidet pTG1162 fås ved kloning af Pstl-Pstl-fragmentet fra fagen M13TG190 i S Pstl-stedet på plasmidet pTGl86-POLY.Plasmid pTG1162 is obtained by cloning the Pstl-Pstl fragment from phage M13TG190 at the S Pstl site of plasmid pTGl86-POLY.
EKSEMPEL 19 10 Konstruktion af plasmidet PTG1163EXAMPLE 19 Construction of the plasmid PTG1163
Det synes også at være af betydning at opnå et rekombinant vacciniavirus, der syntetiserer et ikke-spaltet gpl60, som udskilles i mediet. Dette protein ville i realiteten kunne anvendes som dræbt vaccine i forbindelse med adjuvanser eller 15 inkorporeret i liposomer eller ISCOMS (Morein et al., Nature (1984) 308. 5958, s. 457-460).It also appears to be important to obtain a recombinant vaccinia virus that synthesizes a non-cleaved gp160 that is secreted into the medium. This protein could in fact be used as a killed vaccine for adjuvants or incorporated into liposomes or ISCOMS (Morein et al., Nature (1984) 308. 5958, pp. 457-460).
Til dette formål konstrueres bakteriofagen M13TG194, i hvilken de sekvenser, der koder for de ekstracytoplasmiske og intracytoplasmiske regioner, er fusioneret i 20 fase i bakteriofagen M13TG184 ved hjælp af følgende oligonukleotid: 5' TCCCTGCCTAACTCTATTTI I IATATACCACAGCCA 3'To this end, the bacteriophage M13TG194 is constructed in which the sequences encoding the extracytoplasmic and intracytoplasmic regions are fused in phase 20 in the bacteriophage M13TG184 by the following oligonucleotide: 5 'TCCCTGCCTAACTCTATTTI I IATATACCACAGCA
Pstl-Pstl-fragmentet i M13TG194 klones dernæst i Pstl-stedet på pTG186-POLY, 25 hvilket giver pTGl 163.The PstI-PstI fragment of M13TG194 is then cloned into the PstI site of pTG186-POLY, yielding pTG1163.
De således vundne rekombinante proteiner og især det ikke-spaltelige gpl60 kan anvendes i diagnostiske kits til detektion af potentielle antistoffer, der er til stede i blod fra patienter, som har været i kontakt med viruset. Disse tests kan udføres 30 ved fremgangsmåder, der er kendte for fagfolk, fx ved ELISA, RIPA, "western Blot" (immun-aftryk).The recombinant proteins thus obtained, and in particular the non-cleavable gp160, can be used in diagnostic kits to detect potential antibodies present in the blood of patients who have been in contact with the virus. These tests may be performed by methods known to those skilled in the art, for example, by ELISA, RIPA, "Western Blot" (immune imprint).
Disse proteiner kan også anvendes til fremstilling af hybridomer og monoklonale antistoffer beregnet til detektion af tilstedeværelsen af virus i prøver.These proteins can also be used to prepare hybridomas and monoclonal antibodies designed to detect the presence of virus in samples.
3535
I DK 175613 B1 II DK 175613 B1 I
I 32 II 32 I
I De forskellige plasmider og M13-fager er især beskrevet i følgende publikationer og II The various plasmids and M13 phages are particularly described in the following publications and I
I patentansøgninger: IIn patent applications: I
I M13TG131: Kieny et al., 1983 IIn M13TG131: Kieny et al., 1983 I
I 5 M13TGRG151: WO 83/04052 II 5 M13TGRG151: WO 83/04052 I
I pTG155 PRO: FR 84 06499 II pTG155 PRO: FR 84 06499 I
I M13TG130: Kieny et al., 1983. IIn M13TG130: Kieny et al., 1983. I
I Følgende plasmider blev den 16. november 1984 deponeret i Collection Nationale IIn the following plasmids, November 16, 1984 was deposited in Collection National I
I 10 de Cultures de Microorganismes de l'Ihstitut Pasteur og er beskrevet i GB 84 IIn 10 the Cultures de Microorganisms de l'Ihstitut Pasteur and are described in GB 84 I
I 29099: II 29099: I
I PJ 19-6: CNCM nr. 366-1 IIn PJ 19-6: CNCM No. 366-1 I
I PJ 19-13: CNCM nr. 367-1. IIn PJ 19-13: CNCM No. 367-1. IN
I 15 II 15 I
Plasmidet pTGH25 blev den 6. juni 1986 deponeret i samme institution i form af IThe plasmid pTGH25 was deposited on June 6, 1986 in the same institution in the form of I
transformeret bakterie: Itransformed bacterium: I
I E. colt 1106/pTG1125 med nummeret 1-557. IIn E. colt 1106 / pTG1125 with the number 1-557. IN
I 20 II 20 I
I REFERENCERIN REFERENCES
1. Drillien, R., Spehner, D. (1983) Virology 131, 385-393.1. Drillien, R., Spehner, D. (1983) Virology 131, 385-393.
I 2. Kieny, M.P., Lathe, R. og Lecocq, J.P. 1983. New versatile cloning and 25 sequencing vectors based on bacteriophage M13. Gene 26 :91-99.In 2. Kieny, M.P., Lathe, R. and Lecocq, J.P. 1983. New versatile cloning and 25 sequencing vectors based on bacteriophage M13. Gene 26: 91-99.
3. Kieny, M.P., Lathe, R., Drillieh, R., Spehner, D., Skory, S.,Schmitt, D., I Wiktor, T., Koprowski, H. og Lecocq, J.P. 1984. Expression of rabies virus I glycoprotein from a recombinant vac-cinia virus. Nature 312 : 163-166.3. Kieny, M.P., Lathe, R., Drillieh, R., Spehner, D., Skory, S., Schmitt, D., I Wiktor, T., Koprowski, H. and Lecocq, J.P. 1984. Expression of rabies virus I glycoprotein from a recombinant vaccinia virus. Nature 312: 163-166.
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Claims (30)
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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FR8605043 | 1986-04-08 | ||
FR8605043A FR2596771B1 (en) | 1986-04-08 | 1986-04-08 | VIRAL VECTOR AND RECOMBINANT DNA ENCODING VIA GLYCOPROTEIN CAUSAL AGENT OF THE S.I.D.A, CELL CULTURE INFECTED WITH THIS VECTOR, PROCESS FOR PREPARING GLYCOPROTEIN, GLYCOPROTEIN OBTAINED, VACCINE AND ANTIBODY OBTAINED |
FR8615106 | 1986-10-29 | ||
FR8615106A FR2606029B2 (en) | 1986-04-08 | 1986-10-29 | VIRAL VECTOR AND RECOMBINANT DNA ENCODING VIA GLYCOPROTEIN CAUSAL AGENT OF THE S.I.D.A, CELL CULTURE INFECTED WITH THIS VECTOR, PROCESS FOR PREPARING GLYCOPROTEIN, GLYCOPROTEIN OBTAINED, VACCINE AND ANTIBODY OBTAINED |
FR8700116 | 1987-01-08 | ||
PCT/FR1987/000116 WO1987006260A1 (en) | 1986-04-08 | 1987-04-08 | Viral vector coding for a glycoprotein of the virus responsible for a.i.d.s., vaccine and antibody |
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DK641787A DK641787A (en) | 1987-12-07 |
DK641787D0 DK641787D0 (en) | 1987-12-07 |
DK175613B1 true DK175613B1 (en) | 2004-12-27 |
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DK198706417A DK175613B1 (en) | 1986-04-08 | 1987-12-07 | Virus vector encoding a glycoprotein from the virus responsible for AIDS, vaccine comprising the virus vector or glycoprotein, and antibody to the glycoprotein |
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JP (3) | JP2719917B2 (en) |
KR (1) | KR960001819B1 (en) |
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AU (1) | AU604696B2 (en) |
CA (1) | CA1341353C (en) |
DE (1) | DE3789400T2 (en) |
DK (1) | DK175613B1 (en) |
ES (1) | ES2052589T3 (en) |
FR (1) | FR2606029B2 (en) |
PT (1) | PT84640B (en) |
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US5681713A (en) * | 1987-05-08 | 1997-10-28 | Smithkline Beecham Corporation | Expression of heterologous proteins in Drosophila cells |
US6133029A (en) * | 1988-03-21 | 2000-10-17 | Chiron Corporation | Replication defective viral vectors for infecting human cells |
IL89567A0 (en) * | 1988-03-28 | 1989-09-10 | Univ Leland Stanford Junior | Mutated hiv envelope protein |
US5747324A (en) * | 1988-06-10 | 1998-05-05 | Therion Biologics Corporation | Self-assembled, defective, non-self-propagating lentivirus particles |
WO1989012095A1 (en) * | 1988-06-10 | 1989-12-14 | Applied Biotechnology, Inc. | A method of evaluating recombinant vaccines against immunodeficiency virus |
US5614404A (en) * | 1988-06-10 | 1997-03-25 | Theriod Biologics, Incorporated | Self-assembled, defective, non-self-propagating lentivirus particles |
DE69010523D1 (en) * | 1989-03-03 | 1994-08-18 | Microgenesys Inc | Kit or composition for preventing or treating HIV-1 infections. |
EP0497922B1 (en) * | 1989-10-24 | 2002-01-30 | Chiron Corporation | Infective protein delivery system |
US5817491A (en) * | 1990-09-21 | 1998-10-06 | The Regents Of The University Of California | VSV G pseusdotyped retroviral vectors |
US6228608B1 (en) * | 1991-02-28 | 2001-05-08 | Aquila Biopharmaceuticals, Inc. | Recombinant FIV glycoprotein 160 and P24 gag protein |
FR2676071B1 (en) * | 1991-05-02 | 1994-11-18 | Transgene Sa | NEW NON-CLeavABLE, SOLUBLE, HYBRID-FORMING GP160 VARIANT. |
GB9215233D0 (en) * | 1992-07-17 | 1992-09-02 | Pitman Moore Inc | Vaccines |
US5888814A (en) * | 1994-06-06 | 1999-03-30 | Chiron Corporation | Recombinant host cells encoding TNF proteins |
FR2766091A1 (en) * | 1997-07-18 | 1999-01-22 | Transgene Sa | ANTITUMOR COMPOSITION BASED ON MODIFIED IMMUNOGENIC POLYPEPTIDE WITH CELL LOCATION |
CA2531773A1 (en) | 2003-07-21 | 2005-02-17 | Transgene S.A. | Novel multifunctional cytokines |
CN1304580C (en) * | 2004-07-09 | 2007-03-14 | 楼伟 | Surface glucoprotein gp160 of recombination expression human acquired immunity defact virus 1 |
WO2014009433A1 (en) | 2012-07-10 | 2014-01-16 | Transgene Sa | Mycobacterium resuscitation promoting factor for use as adjuvant |
EP2912069B1 (en) | 2012-10-23 | 2019-07-31 | Emory University | Gm-csf and il-4 conjugates, compositions, and methods related thereto |
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ZA867281B (en) * | 1985-09-25 | 1987-05-27 | Oncogen | Vaccines and immunoassays for acquired immune deficiency syndrome |
IL82104A0 (en) * | 1986-04-08 | 1987-10-30 | Usa | Recombinant vaccinia virus expressing human retrovirus genes and method for producing htlb-iii envelope proteins |
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1986
- 1986-10-29 FR FR8615106A patent/FR2606029B2/en not_active Expired
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1987
- 1987-04-08 EP EP87400790A patent/EP0245136B1/en not_active Expired - Lifetime
- 1987-04-08 AU AU72349/87A patent/AU604696B2/en not_active Expired
- 1987-04-08 WO PCT/FR1987/000116 patent/WO1987006260A1/en unknown
- 1987-04-08 CA CA000534170A patent/CA1341353C/en not_active Expired - Lifetime
- 1987-04-08 AT AT87400790T patent/ATE103328T1/en not_active IP Right Cessation
- 1987-04-08 ES ES87400790T patent/ES2052589T3/en not_active Expired - Lifetime
- 1987-04-08 PT PT84640A patent/PT84640B/en unknown
- 1987-04-08 DE DE3789400T patent/DE3789400T2/en not_active Expired - Lifetime
- 1987-04-08 JP JP62502264A patent/JP2719917B2/en not_active Expired - Lifetime
- 1987-12-07 DK DK198706417A patent/DK175613B1/en active
- 1987-12-08 KR KR87701159A patent/KR960001819B1/en not_active IP Right Cessation
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1995
- 1995-10-25 JP JP7277533A patent/JP2743164B2/en not_active Expired - Lifetime
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Also Published As
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DK641787A (en) | 1987-12-07 |
EP0245136A1 (en) | 1987-11-11 |
ES2052589T3 (en) | 1994-07-16 |
DK641787D0 (en) | 1987-12-07 |
PT84640A (en) | 1987-05-01 |
AU604696B2 (en) | 1991-01-03 |
JPH01500161A (en) | 1989-01-26 |
KR880701283A (en) | 1988-07-26 |
JP2719917B2 (en) | 1998-02-25 |
AU7234987A (en) | 1987-11-09 |
DE3789400D1 (en) | 1994-04-28 |
JPH08224090A (en) | 1996-09-03 |
JPH1059868A (en) | 1998-03-03 |
WO1987006260A1 (en) | 1987-10-22 |
FR2606029A2 (en) | 1988-05-06 |
DE3789400T2 (en) | 1994-09-01 |
CA1341353C (en) | 2002-04-02 |
ATE103328T1 (en) | 1994-04-15 |
JP2743164B2 (en) | 1998-04-22 |
PT84640B (en) | 1989-11-30 |
KR960001819B1 (en) | 1996-02-05 |
EP0245136B1 (en) | 1994-03-23 |
FR2606029B2 (en) | 1989-02-03 |
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