NL8901612A - Vaccines against meningococcal infections - contg. outer membrane proteins or fragments - Google Patents
Vaccines against meningococcal infections - contg. outer membrane proteins or fragments Download PDFInfo
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- 208000034762 Meningococcal Infections Diseases 0.000 title abstract 2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/255—Salmonella (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6068—Other bacterial proteins, e.g. OMP
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
Description
N.O. 36017N.O. 36017
Multivalent meningococcen klasse 1 buitenroembraaneiwit vaccin.Multivalent meningococcal class 1 outer membrane protein vaccine.
De uitvinding heeft betrekking op vaccins welke gebaseerd zijn op meningococcen buitenmembraaneiwitten. Meer in het bijzonder heeft de uitvinding betrekking op vaccins en methoden van produktie gericht op de zgn. klasse 1 meningococcen buitenmembraaneiwitten en daarvan afgeleide peptiden. Genoemde eiwitten resp. peptiden kunnen geproduceerd worden door middel van nieuwe produktiestammen, -methoden, recombinant DNA methoden of peptidesyntheses.The invention relates to vaccines based on meningococcal outer membrane proteins. More particularly, the invention relates to vaccines and methods of production aimed at the so-called class 1 meningococcal outer membrane proteins and peptides derived therefrom. Said proteins resp. peptides can be produced by new production strains, methods, recombinant DNA methods or peptide syntheses.
Meningococcen-ziekte wordt vrijwel uitsluitend veroorzaakt door serogroep A,B,C,W-135,Y meningococcen. Ter voorkoming van deze ziekte kan een tetravalent A,C,W-135,Y-polysaccharide vaccin worden toegepast. Dit vaccin bezit de aan polysacchariden inherente immunologische eigenschappen, zoals een slechte immunogeniteit bij jonge kinderen en geen inductie van het immunologisch geheugen bij individuen, die niet eerder met de desbetreffende bacterie in aanraking zijn geweest. Dientengevolge worden de polysaccharide-vaceins slechts bij risicogroepen zoals militairen en reizigers naar gebieden met een hoge incidentie aan meningococcen-ziekte alsook tijdens verheffingen van de ziekte toegepast.Meningococcal disease is caused almost exclusively by serogroup A, B, C, W-135, Y meningococci. A tetravalent A, C, W-135, Y polysaccharide vaccine can be used to prevent this disease. This vaccine has the immunological properties inherent in polysaccharides, such as poor immunogenicity in young children and no induction of the immunological memory in individuals who have not previously had contact with the bacterium in question. As a result, the polysaccharide vases are used only in at-risk groups such as military personnel and travelers to areas with a high incidence of meningococcal disease as well as during elevations of the disease.
Een groot probleem ten aanzien van de ontwikkeling van een geschikt polysaccharide vaccin tegen meningococcen-ziekte is gelegen in de zeer geringe immunogeniteit van het groep B polysaccharide. Een mogelijke verklaring hiervoor is gelegen in de grote overeenkomst in struktuur tussen het meningococcen groep B polysaccharide en humane glycolipiden. Daar groep B meningococcen in vele landen meer dan 50% van de gevallen van meningococcen-ziekte veroorzaken (Poolman et al·., 1986, Lancet,A major problem with regard to the development of a suitable polysaccharide vaccine against meningococcal disease lies in the very low immunogenicity of the group B polysaccharide. A possible explanation for this lies in the great similarity in structure between the meningococcal group B polysaccharide and human glycolipids. Since group B causes meningococci in many countries more than 50% of cases of meningococcal disease (Poolman et al., 1986, Lancet,
Sept. blz. 555-558) biedt de toepassing van het bovengenoemde A,C,W-135,Y poiysaccharide-vaccin geen soelaas. Het is nog wel mogelijk de immunogeniteit van het B polysaccharide sterk te verhogen door chemische modificatie, maar hierbij rijst de vraag in hoeverre een der— gelijk gemodificeerd groep B polysaccharide na vaccinatie aanleiding zal geven tot ongewenste nevenreacties ten gevolge van een autoimmuün-respons.Sept. pp. 555-558) the use of the aforementioned A, C, W-135, Y polyaccharide vaccine does not offer any relief. It is still possible to strongly increase the immunogenicity of the B polysaccharide by chemical modification, but the question arises to what extent such a modified group B polysaccharide will give rise to undesired side reactions as a result of an autoimmune response after vaccination.
De groep A,C,W-135.Y polysaccharide-vaccins zouden sterk aan werkzaamheid toenemen, indien de polysacchariden chemisch gekoppeld werden aan eiwit. Dit zou de toepasbaarheid ervan bij jonge kinderen, de leeftijdsgroep met de meeste gevallen van meningococcen-ziekte, zeer waarschijnlijk maken, Ten aanzien van het' groep B polysaccharide zijn meningococcan-eiwitten zelfs mogelijk een uitweg ten aanzien van de toepassing van vaccins ter voorkoming van groep B meningocoeeen-ziekte.The group A, C, W-135.Y polysaccharide vaccines would greatly increase in efficacy if the polysaccharides were chemically coupled to protein. This would make it very likely to be useful in young children, the age group with the most cases of meningococcal disease. With regard to the group B polysaccharide, meningococcan proteins may even be a way out of the use of vaccines to prevent group B meningocoeic disease.
Om bovengenoemde redenen is derhalve getracht buitenmembraaneiwitten van meningococcen tot vaccinkomponent te ontwikkelen, zowel als dragereiwit c.q. additionele vaccinkomponent bij A,C,W-I35,Y pólysaecharide-vaccins als primair groep B meningococcen-vaccin. Meningococcen bezitten echter vele buitenmembraaneiwitten, waarvan een tweetal type eiwitten door Aanvraagster zijn geselekteerd voor vacinontwikkeling: klasse 1 en klasse 2/3 buitenmembraaneiwitten (Frasch et al·., 1985, Rev. Infect. Dis.7: blz. 504-510). Hiervan afgeleide buitenmembraaneiwit-vaccins bevatten gezuiverde klasse 1 en 2/3 buitenmembraaneiwitten van een stam, waarbij het endotoxine-gehalte tot een zo laag mogelijke Waarde is teruggebracht. Dergelijke eerste generatie buitenmembraaneiwit-vaccins zijn door verschillende onderzoekers ontwikkeld en vormen de basis voor een aantal octrooien (EP-A-0 145 359; EP-A-0 109 668; EP-A-Q 088 303). Het bezwaar van dergelijke, eerste generatie buitenmembraaneiwit vaccins is gelegen in de stamafhankelijke immuniteit, welke hiermede kan worden opgewekt. Er komen namelijk binnen het species Nelsseria meningitidis (meningococcen) meerdere klasse 1 en 2/3 buitenmembraaneiwitten voor.For the above reasons, it has therefore been attempted to develop outer membrane proteins of meningococci into a vaccine component, both as carrier protein or additional vaccine component in A, C, W-I35, Y polysaecharide vaccines as primary group B meningococcal vaccine. Meningococci, however, possess many outer membrane proteins, of which two types of proteins have been selected by the Applicant for vaccine development: class 1 and class 2/3 outer membrane proteins (Frasch et al., 1985, Rev. Infect. Dis. 7: 504-510). Outer membrane protein vaccines derived therefrom contain purified class 1 and 2/3 outer membrane proteins of a strain, with the endotoxin content reduced to the lowest possible value. Such first generation outer membrane protein vaccines have been developed by various researchers and form the basis for a number of patents (EP-A-0 145 359; EP-A-0 109 668; EP-A-Q 088 303). The drawback of such first-generation outer membrane protein vaccines resides in the strain-dependent immunity which can be raised with them. Namely, there are several class 1 and 2/3 outer membrane proteins within the species Nelsseria meningitidis (meningococci).
Deze uitvinding is gerelateerd aan de wenselijkheid stamonafhanke-lijke immuniteit* op te wekken tegen meningococcen zodat een algemene vaccinatie tegen meningococcen-ziekte kan worden uitgevoerd. Het is gebleken, dat vooral het klasse 1 meningococcen buitenmembraaneiwit bactericide, proefdier beschermende immuniteit kan opwekken (Poolman et al., 1987, Ant.v.Leeuwenh. 53: blz. 413-419; Saukkonen et al», 1987, Mierob. Pathogen.3: blz. 261-267). Het epidemiologische onderzoek laat echter zien, dat met ongeveer tien monoclonale antistoffen tegen het meningococcen klasse 1 buitenmembraaneiwit vrijwel alle stammen zijn te typeren. (Abdillahl and Poolman, 1988, Microb. Pathogen.4: blz. 27-32). Tevens is gebleken, dat een meningococcen-stam twee onafhankelijke klasse 1 buitenmembraaneiwit specifieke monoclonale antistoffen kan binden. Deze bindingsplaatsen, ook wel epitopen genoemd, zijn beiden doelwit voor bactericide antistoffen, zodat ieder klasse 1 buitenmembraaneiwit twee onafhankelijke aangrijpingspunten, geschikt voor vaccinontwikkeiing, bevat. Het kombineren van vijf of meer klasse 1 buitenmembraaneiwitten, bevattende tien of meer doelwit-epitopen, tot een vaccin is echter met zuiveringstechieken niet mogelijk vanwege het meezuiveren van endotoxinen. Tevens worden minder relevante eiwitten meegezuiverd.This invention is related to the desirability of inducing stem-independent immunity * against meningococci so that a general vaccination against meningococcal disease can be performed. It has been shown that especially the class 1 meningococcal outer membrane protein can induce bactericidal, animal protective immunity (Poolman et al., 1987, Ant.v. Leeuwenh. 53: 413-419; Saukkonen et al., 1987, Mierob. Pathogen 3: 261-267). The epidemiological study shows, however, that with almost ten monoclonal antibodies against the meningococcal class 1 outer membrane protein, almost all strains can be typified. (Abdillahl and Poolman, 1988, Microb. Pathogen. 4: 27-32). It has also been shown that a meningococcal strain can bind two independent class 1 outer membrane protein specific monoclonal antibodies. These binding sites, also called epitopes, are both targets for bactericidal antibodies, so that each class 1 outer membrane protein contains two independent targets, suitable for vaccine development. However, combining five or more class 1 outer membrane proteins, containing ten or more target epitopes, into a vaccine is not possible with purification techniques due to the co-purification of endotoxins. Less relevant proteins are also purified.
Gevonden werd, dat de bovengenoemde nadelen kunnen worden opgeheven door het op zinvolïe wijze verbeteren van de produktiemethoden van meningococcen buitenmembraaneiwit vaccins, toegespitst op het bereiden van een multivalent klasse 1 buitenmembraaneiwit vaccin.It has been found that the above drawbacks can be overcome by meaningfully improving the production methods of meningococcal outer membrane protein vaccines focused on preparing a multivalent class 1 outer membrane protein vaccine.
De uitvinding heeft betrekking op vaccins, welke gerelateerd zijn aan - het bereiden van fragmenten van het klasse 1 buitenmembraaneiwit, bijv. door middel van cyanogeen bromide; - het bereiden van zuivere klasse 1 buitenmembraaneiwitten, uitgaande van mutant stammen, welke het klasse 2/3 buitenmembraaneiwit niet tot expressie brengen; - het bereiden van zuivere klasse 1 buitenmembraaneiwitten met behulp van gekloneerd DNA in produktievectoren. Dit omvat ook de toepassing van protein-engineerlng met het doel meerdere epitopen in een DNA leesframe te manipuleren en te produceren; en - het bereiden van een multivalent klasse 1 buitenmerabraaneiwit vaccin door middel van peptide-synthese, aangezien de epitopen met een korte peptideketen synthetisch bereid kunnen worden.The invention relates to vaccines which are related to - preparing fragments of the class 1 outer membrane protein, e.g. by means of cyanogen bromide; - preparing pure class 1 outer membrane proteins, starting from mutant strains, which do not express the class 2/3 outer membrane protein; - preparing pure class 1 outer membrane proteins using cloned DNA in production vectors. This also includes the use of protein engineering for the purpose of manipulating and producing multiple epitopes in a DNA reading frame; and - preparing a multivalent class 1 outer marrow protein vaccine by peptide synthesis, since the epitopes with a short peptide chain can be synthetically prepared.
Gebleken is, dat meningococcen klasse 1 buitenmembraaneiwitten een krachtige bactericide immuunrespons opwekken tegen de juiste epitopen bevattende stammen, ongeacht of dit groep A,B,C,W~135,Y meningococcen zijn. Dit impliceert, dat het A,C,W-135,Y polysacchartde-vaccin vervangen kan worden door een vaccin volgens de uitvinding, dat als breedwerkend meningococcen-vaccin toegepast kan worden. De door het klasse 1 buitenmembraaneiwit opgewekte bactericide antistoffen reageren krachtig met afgesplitste fragmenten en korte synthetische peptiden bereid uitgaande van de aminozuurvolgorde van klasse 1 buitenmembraaneiwitten. Gezien het gegeven dat meningococcen-ziekte tegenwoordig hoofdzakelijk door- groep B meningococcen wordt veroorzaakt alsook het feit, dat de groep B meningococcen klasse 1 buitenmembraaneiwitten ook bij groep A, C,W-135,Y meningococcen voorkomen, heeft de uitvinding met voordeel betrekking op een vaccin, dat door een of meer fragmenten van groep B meningococcen klasse 1 buitenmembraaneiwitten is gekenmerkt. Bij voorkeur wordt bij de bereiding van een dergelijk vaccin van ten minste tien epitopen uitgegaan, welke geselekteerd zijn op epidemiologische gronden. De vaccins volgens de uitvinding bevatten bijvoorbeeld 10 eiwitten verkregen door zuivering uit mutant stammen, of 10 fragmenten bereid door cyanogeenbromide-fragmentatie, of 10 synthetische peptiden, representatief voor 10 epitopen, of produkten verkregen uit via recombinant DNA technologie verkregen genprodukten bevattende de 10 gewenste epitopen. Voorts kunnen de vaccins volgens de uitvinding met voordeel meningococcen A en C of eventueel W-135 en Y polysacchariden en/of detergentia bevatten. Bij voorkeur worden de A en C polysacchariden covalent gekoppeld aan de eiwitprodukten. Detergentia kunnen zowel zwitterionogene, kationogene, anionogene en niet-ionogene detergentia zijn. Voorbeelden van dergelijke detergentia zijn Zwlttergent 3-10, Zwittergent 3-14 (N-tetradecyl-N,N-diaethyl-3-ammonia-l-propane sulfona-te), Tween-20, natriunr-desoxycholaat, natriumcholaat en octylglueoside. Tevens kan het vaccin volgens de uitvinding een adsorbent zoals aluminiumhydroxide, calciumfosfaat of met voordeel aluminiutafosfaat bevatten. De fragmenten, eiwitten, peptiden kunnen ook in immunostiffiu-lerende complexen (Isocoms), liposomen of door middel van andere adju-vantia worden verwerkt, zodat grotere immunogeniteit wordt verkregen.Meningococcal class 1 outer membrane proteins have been shown to elicit a potent bactericidal immune response against the appropriate epitope-containing strains, regardless of whether they are group A, B, C, W ~ 135, Y meningococci. This implies that the A, C, W-135, Y polysacchartde vaccine can be replaced by a vaccine according to the invention, which can be used as a broad-acting meningococcal vaccine. The bactericidal antibodies raised by the class 1 outer membrane protein react vigorously with cleaved fragments and short synthetic peptides prepared from the amino acid sequence of class 1 outer membrane proteins. The invention advantageously relates to the fact that meningococcal disease is currently mainly caused by group B meningococci and that the group B meningococcal class 1 outer membrane proteins also occur in groups A, C, W-135, Y meningococci. a vaccine, characterized by one or more fragments of group B meningococcal class 1 outer membrane proteins. Preferably, the preparation of such a vaccine is based on at least ten epitopes selected on epidemiological grounds. For example, the vaccines of the invention contain 10 proteins obtained by purification from mutant strains, or 10 fragments prepared by cyanogen bromide fragmentation, or 10 synthetic peptides, representative of 10 epitopes, or products obtained from gene products obtained by recombinant DNA technology containing the 10 desired epitopes . Furthermore, the vaccines according to the invention may advantageously contain meningococci A and C or optionally W-135 and Y polysaccharides and / or detergents. Preferably, the A and C polysaccharides are covalently coupled to the protein products. Detergents can be zwitterionic, cationic, anionic and nonionic detergents. Examples of such detergents are Zwltertergent 3-10, Zwittergentgent 3-14 (N-tetradecyl-N, N-diaethyl-3-ammonia-1-propane sulfonate), Tween-20, sodium r-deoxycholate, sodium cholate and octylglueoside. The vaccine according to the invention can also contain an adsorbent such as aluminum hydroxide, calcium phosphate or advantageously aluminum phosphate. The fragments, proteins, peptides can also be processed in immunostiffulatory complexes (Isocoms), liposomes or by other adjuvants, so as to obtain greater immunogenicity.
BESCHRIJVING RESULTATENDESCRIPTION RESULTS
Typespecifieke monoclonale antistoffen werden bereid tegen verschillende meningococcen klasse 1 buitenmembraaneiwitten. Deze raonoclo-nale antistoffen omvatten de volgende subtypen: Pl.l, P1.2, PI.6, PI.7, PI.9, PI,10, PI.15, Pl.16, Pl.17. Al deze monoclonale antistoffen reageren met het SDS (sodium dodecyl sulfaat) gedenatureerde eiwit indien getest met Western blotting. Tevens bleek dat een aantal van deze monoclonale antistoffen reageerde met een 25Kd CNBr (cyanogeenbromide) fragment van het 42Kd klasse 1 buitenmembraaneiwit. Dit resultaat impliceerde, dat de klasse 1 buitenmembraaneiwit epitopen hoofdzakelijk van het lineaire type zijn en zodoende met synthetische peptiden zijn na te bootsen. De epidemiologische resultaten van door Aanvraagster uitgevoerde proeven tonen dat beschreven monoclonale antistoffen het merendeel van de groep A,B,C meningococcen kan typeren hetgeen een beperkte heterogeniteit suggereert. Tevens blijkt dat een klasse l buitenmembraaneiwit twee afzonderlijke typespecifieke epitopen bevat (Abdillahi and Poolman, Microb. Pathogen. 1988, 4: blz, 27-32; idem FEMS Microbiol. Immunol. 47: blz. 139-144).Type-specific monoclonal antibodies were prepared against various meningococcal class 1 outer membrane proteins. These ronaloclonal antibodies include the following subtypes: Pl.1, P1.2, PI.6, PI.7, PI.9, PI, 10, PI.15, Pl.16, Pl.17. All of these monoclonal antibodies react with the SDS (sodium dodecyl sulfate) denatured protein when tested with Western blotting. It was also found that some of these monoclonal antibodies reacted with a 25Kd CNBr (cyanogen bromide) fragment of the 42Kd class 1 outer membrane protein. This result implied that the class 1 outer membrane protein epitopes are mainly of the linear type and thus mimic with synthetic peptides. The epidemiological results of tests conducted by the Applicant show that the described monoclonal antibodies can typify the majority of the group A, B, C meningococci, suggesting a limited heterogeneity. It also appears that a class 1 outer membrane protein contains two separate type-specific epitopes (Abdillahi and Poolman, Microb. Pathogen. 1988, 4: p. 27-32; ditto FEMS Microbiol. Immunol. 47: 139-144).
Het gezuiverde klasse 1 buitenmembraaneiwit, subtype PI.7,16, afkomstig uit de kweek van een klasse 2/3 vrije mutant bleek in een dosering van 2,5/ug bij muizen een bactericide antistoffenrespons op te wekken van 1:64 serumverdunning. De monoclonale antistoffen tegen meningococcen klasse 1 buitenmembraaneiwitten, klasse 2/3 buitenmembraaneiwit ten en lipopolysacchariden werden vergeleken op bactericide werking. De monoclonale antistoffen tegen de klasse 1 buitenmembraan-eiwitten blijken de sterkst bactericide aktiviteit te bezitten (zie Tabel A). De bactericide aktiviteit van deze monoclonale antistoffen blijkt goed te korreleren met de in vivo beschermende aktiviteit, zoals gemeten in een rat meningitis model (Saukkonen et al. 1987, Microbiol.The purified class 1 outer membrane protein, subtype PI.7.16, from the culture of a class 2/3 free mutant at a dose of 2.5 µg in mice was found to elicit a bactericidal antibody response of 1:64 serum dilution. The monoclonal antibodies against meningococcal class 1 outer membrane proteins, class 2/3 outer membrane proteins and lipopolysaccharides were compared for bactericidal activity. The monoclonal antibodies against the class 1 outer membrane proteins appear to have the strongest bactericidal activity (see Table A). The bactericidal activity of these monoclonal antibodies appears to correlate well with the in vivo protective activity, as measured in a rat meningitis model (Saukkonen et al. 1987, Microbiol.
Pathogen. 3: blz. 261-267). Het PI.16 epitoop blijkt aanwezig te zijn op het C-terminalë CNBr fragment van het klasse 1 buitenmembraaneiwit van stam H44/76 (B: 15: PI.7,16). Verdere karakterisering van het PI. 16 epitoop is verricht door middel van bepaling van de aminozuurvolgorde van de 17Kd (N-terminaal) en 25 Kd (C-terminaal) CNBr fragmenten. Het Oterminaal 25Kd is verder gefragmenteerd met V8 protease, endoLysC, endoGlu-C en endoArg-C. Met het PI.16 monoclonale antilichaam positieve fragmenten zijn gesequenced voor zover mogelijk en deze fragmenten zijn vervolgens nagebootst met synthetische peptiden. De bepaalde sequenties zijn als volgt: N-terminus: DVSLYGEIKAGVEDRNYQLQLTEAQUAANG N-terminus 25Kd C-terminale CNBrPathogen. 3: 261-267). The PI.16 epitope appears to be present on the C-terminal CNBr fragment of the class 1 outer membrane protein of strain H44 / 76 (B: 15: PI.7,16). Further characterization of the PI. 16 epitope was performed by determining the amino acid sequence of the 17Kd (N-terminal) and 25 Kd (C-terminal) CNBr fragments. The Terminal 25Kd is further fragmented with V8 protease, endoLysC, endoGlu-C and endoArg-C. PI.16 monoclonal antibody positive fragments are sequenced as far as possible and these fragments are then mimicked with synthetic peptides. The determined sequences are as follows: N-terminus: DVSLYGEIKAGVEDRNYQLQLTEAQUAANG N-terminus 25Kd C-terminal CNBr
fragment: (-M) PVSVRYDSPEFSGFSGSVQFVPIONSKSAYTPAYYTKOTNNNfragment: (-M) PVSVRYDSPEFSGFSGSVQFVPIONSKSAYTPAYYTKOTNNN
Met V8 protease en endoArg-C fragmentering werden met P1.L6 monoclonale antiliehamen reagerende peptiden geïsoleerd met mol.wt. van 7-9Kd en 4-6Kd respectievelijk. De N-terminale sequenties hiervan zijn als volgt:V8 protease and endoArg-C fragmentation peptides reacting with P1.L6 monoclonal antibodies were isolated with mol.wt. from 7-9Kd and 4-6Kd respectively. The N-terminal sequences of these are as follows:
V8 7-9Kd fragment: FSGFSGSVQFVP1QNSKSAYTPAYYTKDIN- -RNAFEV8 7-9Kd fragment: FSGFSGSVQFVP1QNSKSAYTPAYYTKDIN- -RNAFE
Arg-C 4-6Kd fragment; PVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKArg-C 4-6Kd fragment; PVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTK
Op grond Van deze resultaten werden een aantal synthetische peptiden bereid waarvan het volgende peptide een duidelijk positieve reaktie met de PI.16 monoclonale antilichamen vertoont: EFSGFSGSVQFVPIQNSKSAYTPAYYTKDTNNNBased on these results, a number of synthetic peptides were prepared, the following peptide of which shows a marked positive reaction with the PI.16 monoclonal antibodies: EFSGFSGSVQFVPIQNSKSAYTPAYYTKDTNNN
Voor onderzoek naar de exacte chemische identiteit van de omschreven epitopen werd de aminozuurvolgorde vastgesteld als afgeleide van de nucleotide volgorde van de strukturele genen van een drietal meningo-coccen klasse 1 buitenmembraaneiwitten met verschillende subtypespecifi-citeit. Vergelijking met een drietal aminozuurvolgorden zal een voorspelling van de exacte epitopen mogelijk maken, hetgeen bevestigd kan worden met behulp van peptide-synthese. De genen Werden gekloneerd in lambda gtll vector (Barlow et al. (1987), Infeet.Immun.55:2743-2740), gesubkloneerd in Ml3 sequencing vectors en de nucleotidevolgorde werd vastgesteld.To investigate the exact chemical identity of the epitopes described, the amino acid sequence was determined as a derivative of the nucleotide sequence of the structural genes of three meningococcal class 1 outer membrane proteins with different subtype specificities. Comparison with three amino acid sequences will allow prediction of the exact epitopes, which can be confirmed by peptide synthesis. The genes were cloned into lambda gtll vector (Barlow et al. (1987), Infeet.Immun.55: 2743-2740), subcloned into Ml3 sequencing vectors and the nucleotide sequence was determined.
De afgeleide aminozuurvolgorde voor PI.7,16;PI.16;P1.15 eiwitten is als volgt: 10 20 30 40 50The deduced amino acid sequence for PI.7,16; PI.16; P1.15 proteins is as follows: 10 20 30 40 50
PI.16 :DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQV—KVTKAKSRIRTKISPI.16: DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQV — KVTKAKSRIRTKIS
**************** * **** ****************************** * **** **************
PI.15 :DVSLYGEIKAGVEGRNFQLQLTEPP-SKSQP~—QV—KVTKAKSR1RTK.ISPI.15: DVSLYGEIKAGVEGRNFQLQLTEPP-SKSQP ~ —QV—KVTKAKSR1RTK.IS
**************** * **** ****************************** * **** **************
PI .7,16: DVSLYGEIK.AGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISPI .7,16: DVSLYGEIK.AGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKIS
60 70 80 90 100 110 DF GSFIGFKGSEDLGE GLKAVWQLEQDVSVAGGGAS QWGNRESFIGLAGEFGTLRAGRVA *************** ******************* ******** *************** dfgsfigfkgsedlgeglkavwqleqdvsvagggatqwgnresfvglagefgtlragrva *************** ******************* ******** *************** DF GSFIGFKGSEDLGDGLKAVWQLEQDVS VAGGGATQWGNRESFIGL.AGEFGTLRAGRVA . *************** ******************* ******** *************** 120 130 140 150 160 170 N QFDDASQAINPWD SNNDVASQLGIFKRHDDMPVSVRYDSPEF SGF SGSV QFVPAQNSKS ********** ****************************** ************ ***** N QF DDASQAIDP WD SNNDVASQLGIFKRHDDMPVSVRYD SPD FS GF SGSVQFVPIQNS KS ********** ****************************** ************ ***** NQFDDASQAIDPWDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKS ********** ****************************** ************ ***** 180 190 200 210 220 230 AYKPAYYTKDTNNNLTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAF ** ** ** * ************************** ************* ** AYTPAflYTRQNNTDV-FVPAVVGKPGSDVYYAGLNYKNGGFAGSYAFKYARHANVGRDAF ** ** ** * ************************** ************* **60 70 80 90 100 110 DF GSFIGFKGSEDLGE GLKAVWQLEQDVSVAGGGAS QWGNRESFIGLAGEFGTLRAGRVA *************** ******************* ****** ** *************** dfgsfigfkgsedlgeglkavwqleqdvsvagggatqwgnresfvglagefgtlragrva *************** ***************** ** ******** *************** DF GSFIGFKGSEDLGDGLKAVWQLEQDVS VAGGGATQWGNRESFIGL.AGEFGTLRAGRVA. *************** ******************* ******** ******** ******* 120 130 140 150 160 170 N QFDDASQAINPWD SNNDVASQLGIFKRHDDMPVSVRYDSPEF SGF SGSV QFVPAQNSKS ********** ********************* ********* ************ ***** N QF DDASQAIDP WD SNNDVASQLGIFKRHDDMPVSVRYD SPD FS GF SGSVQFVPIQNS KS ********** **** ************************** ************ ***** NQFDDASQAIDPWDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKS ****** **** ****************************** ************ **** * 180 190 200 210 220 230 AYKPAYYTKDTNNNLTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAF ** ** ** * ************************* ********* **** ** AYTPAflYTRQNNTDV-FVPAVVGKPGSDVYYAGLNYKNGGFAGSYAFKYARHANVGRDAF ** ** ** * ************************** ******** ***** **
AYTPAYYTKNTNNNLTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFAYTPAYYTKNTNNNLTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAF
240 250 260 270 28Q 290 ELFL I GS AT SD EAKGTDPLKN HQVHRLTGGYEEGGLNLAL AAQLDL SENGD KAKT KNSTT **** ** ** *************************************** *******240 250 260 270 28Q 290 ELFL I GS AT SD EAKGTDPLKN HQVHRLTGGYEEGGLNLAL AAQLDL SENGD KAKT KNSTT **** ** ** ************************* ************** *******
Ej_.F LLGS-T SD E AKGTDPLKKHQVHRLTGGYEE GGL NLALAAQL DL SEN GD KAKT KNSTT **** ** ** *************************************** *******Ej_.F LLGS-T SD E AKGTDPLKKHQVHRLTGGYEE GGL NLALAAQL DL SEN GD KAKT KNSTT **** ** ** ************************** ************* *******
ΕΕΡΕΙΟδ-ΟεΟΟΑΚΰΤΟΡΕΚΝΗΟνΗΡΕΤΟΟΥΕΕΟΰΕΝΕΑΕΑΑςΕΒΕΒΕΝΟΒ—KTKNSTTΕΕΡΕΙΟδ-ΟεΟΟΑΚΰΤΟΡΕΚΝΗΟνΗΡΕΤΟΟΥΕΕΟΰΕΝΕΑΕΑΑςΕΒΕΒΕΝΟΒ — KTKNSTT
300 310 320 330 340 350 EIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAM ************************ *********************************** EIAATAS YRFGNAVPRIS YAtïGFDLIERGKKGENTS YD QIIAGVDYDF SKRTS AIVSGAW ************************ *********************************** EIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVS GAWT ************************ *********************************** 360 370 LKR NTGIGNY TQINAASVGL RHKF ************************ LKRNTGIGNYTQINAASVGLRHKF ** **********************300 310 320 330 340 350 EIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAM ************************ ******************** **************** EIAATAS YRFGNAVPRIS YAtïGFDLIERGKKGENTS YD QIIAGVDYDF SKRTS AIVSGAW ************************ *** ******************************** EIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVS GAWT **************** ******** *********************************** 360 370 LKR NTGIGNY TQINAASVGL RHKF * *********************** LKRNTGIGNYTQINAASVGLRHKF ** **********************
LKRNTGIGNYTQINAASVGLRHKFLKRNTGIGNYTQINAASVGLRHKF
************************************************
Hieruit kan worden afgeleid, dat de volgorde van aminozuren 24-34 en 176-187 sterk verschillend is in de drie eiwitsequenties en naar alle waarschijnlijkheid de twee afzonderlijke epitopen omvatten zoals eerder beschreven op grond van stamtyperingscesultaten, Dit werd bevestigd met behulp van peptidesynthese en reaktie van de peptiden met PI.7, PI.15 en PI.16 specifieke monoclonale antistoffen. Van'overlappende decapeptiden van het PI.16 eiwit met het PI. 16 monoclonale antilichaam reageerde alleen het decapeptide YYTKDTNNNL zoals verwacht. In de PI.7,16 stam is de volgorde YYTKNTNNNL aanwezig, de D-N verandering op plaats 180 heeft weliswaar enig effekt op antistofbinding. De volgorde HYTRQNNTDVF in het PI. 15 op dezelfde plaats in het eiwit als de PI,16 epitoop is verantwoordelijk voor binding aan het PI.15 monoclonale antilichaam. De volgorde van 24-34 van het PI.7,16 eiwit AQAANGGASG is verantwoordelijk voor binding aan het PI.7 monoclonale antilichaam. Het is waarschijnlijk, dat de volgorden QPQVTNGVQGN en PPSKSQP in de Pl.lö en Pl.15 eiwitten ook epitopen vertegenwoordigen, maar daar zijn nog geen monoclonale antilichatnen tegen ontwikkeld. De twee epitopen van de afzonderlijke eiwitten bevinden zich op zgn. surface loops van deze membraaneiwitten. Dërgelijke porine buitenmembraaneiwitten bevatten meer dan twee surface loops. Dit impliceert dat er surface loops zijn welke qua aminozuurvolgorde identiek zijn binnen de verschillende klasse 1 buitenmembraaneiwitten. Dit opent de weg naar het gebruik van gemeenschappelijke peptiden van het klasse 1 buitenmembraaneiwit voor vacclndoeleinden. Meer in het bijzonder wordt in Fig.l een schematisch twee- dimensionaal model van het meningococcen klasse 1 buitenmembraaneiwit PI.16 weergegeven. Dit model omvat een achttal surface-loops, waarbij de eerste en de vierde loop de op grond van stamtyperlngsresultaten aangetoonde type-specifieke epitopen bevatten.From this it can be deduced that the order of amino acids 24-34 and 176-187 is very different in the three protein sequences and is likely to include the two separate epitopes as described previously based on strain typing results. This was confirmed by peptide synthesis and reaction. of the peptides with PI.7, PI.15 and PI.16 specific monoclonal antibodies. Overlapping decapeptides of the PI.16 protein with the PI. 16 monoclonal antibody reacted only the decapeptide YYTKDTNNNL as expected. In the PI.7,16 strain the sequence YYTKNTNNNL is present, although the D-N change at position 180 does have some effect on antibody binding. The order HYTRQNNTDVF in the PI. 15 at the same site in the protein as the PI, 16 epitope is responsible for binding to the PI.15 monoclonal antibody. The 24-34 sequence of the PI.7.16 protein AQAANGGASG is responsible for binding to the PI.7 monoclonal antibody. It is likely that the sequences QPQVTNGVQGN and PPSKSQP in the Pl.lö and Pl.15 proteins also represent epitopes, but no monoclonal antibodies have yet been developed against them. The two epitopes of the individual proteins are located on so-called surface loops of these membrane proteins. Such porine outer membrane proteins contain more than two surface loops. This implies that there are surface loops that are identical in amino acid sequence within the different class 1 outer membrane proteins. This opens the way for the use of common peptides of the class 1 outer membrane protein for vaccine purposes. More specifically, in Fig. 1, a schematic two-dimensional model of the meningococcal class 1 outer membrane protein PI.16 is shown. This model includes eight surface loops, with the first and fourth loops containing the type-specific epitopes demonstrated by strain typing results.
Een illustratie van de reaktivitelt van het decapeptide YYTKDTNNNL met de PI.16 monoclonale antistof MN5C11G (PI.16 specifiek) wordt in het als Fig,2 opgenomen pep-scan weergegeven.An illustration of the reaction of the decapeptide YYTKDTNNNL with the PI.16 monoclonal antibody MN5C11G (PI.16 specific) is shown in the pep scan included as Fig. 2.
Behalve de epitopen voor de PI.7,16, PI. 16 en PI.15-eiwitten is de epitoop van het P1.2-eiwit door Aanvraagster opgehelderd. Deze epitoop, welke zich op de top van loop 4 bevindt, bezit de samenstelling HFVQQTPQSQP.Except the epitopes for PI.7,16, PI. 16 and PI.15 proteins, the epitope of the P1.2 protein has been elucidated by Applicant. This epitope, which is located at the top of loop 4, has the composition HFVQQTPQSQP.
Behalve op vaccins, gebaseerd op de epitopen, welke karakteristiek zijn voor de bovenvermelde typespecifieke klasse 1 buitenmembraan-eiwitten heeft de uitvinding tevens betrekking op vaccins, gebaseerd op eiwitsegmenten aan de oppervlakte van het membraan, welke volledig of nagenoeg volledig "constant" zijn bij de meningococcen klasse 1 buiten-membraaneiwitten. Met behulp van dergelijke, op "constante" peptiden gebaseerde vaccins kan de stamafhankelijkheid op direkte wijze worden opgeheven. Onder verwijzing naar de bovenvermelde aminozuurvolgorde van de PI.7,16-, PI.16- en P1.15-eiwitten is bij loop 4 (zie mede Fig.l) zowel het peptide AQNSKSAYKPA - dit peptide gaat vooraf aan het typespecifieke epitoop YYTKDTNNNL - als het daarop volgende peptide TLVFAVVGKPG (nagenoeg) constant.In addition to epitope-based vaccines, which are characteristic of the above-mentioned type-specific class 1 outer membrane proteins, the invention also relates to vaccines based on protein segments on the surface of the membrane which are wholly or substantially "constant" at the meningococcal class 1 outer membrane proteins. Using such "constant" peptide-based vaccines, strain dependence can be directly eliminated. Referring to the above amino acid sequence of the PI.7,16, PI.16 and P1.15 proteins in loop 4 (see also Fig. 1), both the peptide AQNSKSAYKPA - this peptide precedes the type-specific epitope YYTKDTNNNL - as the subsequent peptide TLVFAVVGKPG (almost) constant.
Het gebruik van een tiental decapeptiden, bereid via synthese, is geïndiceerd en zeer wel produktietechnisch realiseerbaar. Via recombi-nanttechnieken kunnen meerdere epitopen gekombineerd worden in een lees-frame door middel van fusie-eiwit technologie en/of uitwisseling van stukjes DNA binnen een gen.The use of ten decapeptides, prepared via synthesis, is indicated and is very feasible in production technology. Multiple epitopes can be combined in a reading frame via recombinant techniques by means of fusion protein technology and / or exchange of pieces of DNA within a gene.
BEREIDING VAN EEN MULTIVALENT MENINGOCOCCEN KLASSE I BUITENMEMBRAAN-EIWIT VACCINPREPARATION OF A MULTIVALENT MENINGOCOCCESS CLASS I OUTDOOR MEMBRANE-PROTEIN VACCINE
De bereiding van een multivalent klasse 1 meningococcen buitenmem-braaneiwit vaccin kan op een aantal verschillende manieren worden gerealiseerd. Dit wordt hieronder nader toegelicht; deze toelichting dient niet beperkend te worden uitgelegd.The preparation of a multivalent class 1 meningococcal outer membrane protein vaccine can be accomplished in a number of different ways. This is explained in more detail below; this explanation should not be interpreted restrictively.
METHODE A: Zuivering van klasse 1 buitenmembraaneiwitten uit bacteriologische kweek.METHOD A: Purification of class 1 outer membrane proteins from bacteriological culture.
Deze kweek kan worden verricht met de gewenste wilde type stammen, mutant meningococcen stammen zonder klasse 2/3 buitenmembraaneiwitten en/of homologe en heterologe recombinant micro-organismen welke de gewenste meningococcen klasse 1 buitenmembraaneiwit epitopen tot expressie brengen via overprodueerende vectoren al dan niet via bestaande open reading frames en/of' gemanipuleerde leesframes zodat fusie-eiwitten of eiwitten met verwisselde epitopen kunnen worden bereid.This culture can be performed with the desired wild type strains, mutant meningococcal strains lacking class 2/3 outer membrane proteins and / or homologous and heterologous recombinant microorganisms expressing the desired meningococcal class 1 outer membrane protein epitopes via over-producing vectors, whether or not via existing open reading frames and / or manipulated reading frames so that fusion proteins or proteins with exchanged epitopes can be prepared.
Voorbeelden van wilde type stammen zijn: H44/76(B: 15:P1.7,16) (Holten E, Noorwegen); 187(.3:4:PI .1.7) (Etienne J, Frankrijk.); M1080(B:1 rPl. 1,7) (Frasch· C, USA); Swiss4( B;4:P1.15) (Hirschel B, Zwitserland); B2106(B:4:P1.2); (Berger U, West Duitsland); 395(B;NT:P1.9) (Jonsdottir K, Ijsland); H990(B:6:P1.6) (Frasch C, USA); 2996(B:2b:P1.2) (collectie van aanvraagster); M982(B:9:P1.9) (Frasch C, USA); S3446(B:14:P1.6) (Frasch C, USA); H355(B:15:PI.15) (Holten E, Noorwegen);· 6557( B: 17 ?P1.17) (Zollinger W, USA) en B40(A:4:P1.10) (Achtman M, West Duitsland). Een Voorbeeld van een klasse 3 negatieve mutant is H44/76III5( B.:-:P1,16) (collectie van aanvraagster).Examples of wild type strains are: H44 / 76 (B: 15: P1.7,16) (Holten E, Norway); 187 (.3: 4: PI. 1.7) (Etienne J, France.); M1080 (B: 1 rPl. 1.7) (Frasch · C, USA); Swiss4 (B; 4: P1.15) (Hirschel B, Switzerland); B2106 (B: 4: P1.2); (Berger U, West Germany); 395 (B; NT: P1.9) (Jonsdottir K, Iceland); H990 (B: 6: P1.6) (Frasch C, USA); 2996 (B: 2b: P1.2) (applicant's collection); M982 (B: 9: P1.9) (Frasch C, USA); S3446 (B: 14: P1.6) (Frasch C, USA); H355 (B: 15: PI.15) (Holten E, Norway); 6557 (B: 17? P1.17) (Zollinger W, USA) and B40 (A: 4: P1.10) (Achtman M, West Germany). An Example of a class 3 negative mutant is H44 / 76III5 (B.:-:P1,16) (applicant's collection).
Deze stammen worden van -70°C voorkultures geënt in schudkolven en van daaruit in fermentorkultures van 40, 150 of 350 liter. Het semi-synthetische medium bestaat uit: L-glutaminezuur 1,3 g/1, L-cysteine.HC1 0.02 g/1, Na2HP04.2H20 10 g/l, KC1 0.09 g/1, NaCl 6 g/1, NH4C1 1,25 g/1, MgS04.7H20 0,6 g/1, glucose 5 g/1, Fe(N03)3 100 /uM, gistdialysaat.These strains are inoculated from -70 ° C pre-cultures in shaking flasks and from there into fermenter cultures of 40, 150 or 350 liters. The semi-synthetic medium consists of: L-glutamic acid 1.3 g / 1, L-cysteine. HC1 0.02 g / 1, Na2HP04.2H20 10 g / l, KC1 0.09 g / 1, NaCl 6 g / 1, NH4C1 1 , 25 g / L, MgSO 4 .7H 2 O 0.6 g / L, glucose 5 g / L, Fe (NO 3) 3 100 µM, yeast dialysate.
Gedurende de kweek in de fermentor werden de pH en pÖ2 gecontroleerd en automatisch op een pH van 7,0-7,2 en een luchtverzadiging van 10% geregeld. De cellen werden met behulp van centrifugeren en wassen met steriel 0.1M NaCl geoogst en bij -20° C opgeslagen of gevriesdroogd.During the culture in the fermentor, the pH and pO2 were checked and automatically adjusted to a pH of 7.0-7.2 and an air saturation of 10%. The cells were harvested by centrifugation and washing with sterile 0.1M NaCl and stored or lyophilized at -20 ° C.
De bacteriemassa wordt bijvoorbeeld geëxtraheerd met behulp van 0.5 M CaCl2, 1% (w/v) Zwittergent 3—14 (Zw 3-14) en 0.14 M NaCl, pH 4.0, 100 ml extractievloeistof per gram drooggevroren bacteriemassa. De suspensie wordt gedurende 1 uur bij kamertemperatuur geroerd en vervolgens gecentrifugeerd (1 uur, 3000xg), waarna het supernatant steriel wordt verzameld. Aan het supernatant wordt 20% ethanol toegevoegd (v/v) en na 30 min. roeren wordt het produkt gecentrifugeerd (30 min,, lO.OOOxg) waarna het supernatant aseptisch wordt verzameld. Het supernatant wordt vervolgens door middel van diafiltratie in een Amicon Hollow Fiber System (HlD x 50, cut off 50.000) geconcentreerd en ontdaan van CaCl2 en ethanol. Het concentraat wordt met 0.1M natriumacetaat, 25 mM EDTA, 0.05% Zw 3-14, pH 6.0 tot het oorspronkelijke volume verdund en daarna opnieuw geconcentreerd door middel van.een diafiltratie. Deze procedure wordt vijfmaal herhaald. De pH van het laatste concentraat wordt op een waarde van 4.0 gebracht. Aan het concentraat wordt 20% (v/v) ethanol toegevoegd en na 30 min. roeren wordt het produkt gecentrifugeerd (30 min., LO.OOxg). De hele eiwitten worden gezuiverd met behulp van kolom- chromatografie in aanwezigheid van detergens, bijv. Zw 3-14. Veelal wordt zowel gelfiltratie op Sephacryl S-300 als ionenwisseling op DEAE Sepharose toegepast. De gebruikte extractiemethode, detergentia, kolom-chromatografie zijn niet de enige toe te passen methoden, doch gelden slechts als voorbeelden en dienen niet als beperkend te worden beschouwd.For example, the bacterial mass is extracted using 0.5 M CaCl 2, 1% (w / v) Zwittergent 3-14 (Zw 3-14) and 0.14 M NaCl, pH 4.0, 100 ml extraction liquid per gram of dry frozen bacterial mass. The suspension is stirred at room temperature for 1 hour and then centrifuged (1 hour, 3000xg), after which the supernatant is collected sterile. 20% ethanol is added to the supernatant (v / v) and after stirring for 30 min, the product is centrifuged (30 min, 100,000xg) and the supernatant is collected aseptically. The supernatant is then concentrated by diafiltration in an Amicon Hollow Fiber System (HlD x 50, cut off 50,000) and stripped of CaCl2 and ethanol. The concentrate is diluted to the original volume with 0.1M sodium acetate, 25mM EDTA, 0.05% Zw 3-14, pH 6.0 and then concentrated again by diafiltration. This procedure is repeated five times. The pH of the last concentrate is adjusted to a value of 4.0. 20% (v / v) ethanol is added to the concentrate, and after stirring for 30 min, the product is centrifuged (30 min., LOxg). The whole proteins are purified by column chromatography in the presence of detergent, e.g. Zw 3-14. Often both gel filtration on Sephacryl S-300 and ion exchange on DEAE Sepharose is used. The extraction method, detergents, column chromatography used are not the only methods to be used, but are merely examples and should not be construed as limiting.
METHODE B: Fragmentering van eiwitten.METHOD B: Fragmentation of proteins.
Als eerste methode bij meningoeoccen klasse 1 buitenmembraaneiwitten geldt de cyanogeenbromide-fragmentering. De gezuiverde klasse 1 of mengsels van klasse 1 en 3 buitenmembraanei witten worden opgenomen in 70% mierezuur (v/v) en met 10-voudige overmaat CNBr gedurende 16 uren bij kamertemperatuur behandeld. Het CNBr en mierezuur worden via indam-ping verwijderd en vervangen door 0.2 M tris.HCl, 6 M ureum pH 7.2. Het supernatant wordt door middel van gelfiltratie op Sephacryl S-200 voorgezuiverd en daarna met behulp van TSK-2000 gelfiltratie via HPLC gezuiverd.The first method for meningoeococcal class 1 outer membrane proteins is cyanogen bromide fragmentation. The purified class 1 or mixtures of class 1 and 3 outer membrane proteins are taken up in 70% formic acid (v / v) and treated with 10-fold excess CNBr for 16 hours at room temperature. The CNBr and formic acid are removed by evaporation and replaced with 0.2 M tris.HCl, 6 M urea pH 7.2. The supernatant is pre-purified by gel filtration on Sephacryl S-200 and then purified by TSK-2000 gel filtration via HPLC.
De gezuiverde klasse 1 buitenmembraaneiwitfragmenten kunnen nog verder worden gefragmenteerd met behulp van endoLysC, endoArg-G, endoGlu-C, V8 protease en andere eiwitsplitsende enzymen. Deze fragmenten worden gezuiverd met behulp van HPLC.The purified class 1 outer membrane protein fragments can be fragmented even further using endoLysC, endoArg-G, endoGlu-C, V8 protease and other protein splitting enzymes. These fragments are purified by HPLC.
De eiwitten en/of fragmenten, gezuiverd volgens methoden A en/of B worden tot vaccin verwerkt m.b.v. meningoeoccen C polysaccharide, detergentia, zoals Na-cholaat, Na-desoxycholaat, Tween-20 en andere of door verwerking in liposomen of immunostimulerende complexen. A^CPO^)^ kan als adjuvans worden toegevoegd. Voor de kleinere fragmenten is het nodig dat deeltjesvergroting wordt gerealiseerd en dat ervoor wordt gezorgd, dat voldoende T-help epitopen in het vaccin aanwezig zijn, wat te realiseren is met behulp van toegevoegde homologe of heterologe T-help epitopen.The proteins and / or fragments purified according to methods A and / or B are vaccined using meningococcal C polysaccharide, detergents such as Na cholate, Na deoxycholate, Tween-20 and others or by incorporation into liposomes or immunostimulating complexes. A ^ CPO ^) ^ can be added as an adjuvant. The smaller fragments require that particle enlargement be achieved and that sufficient T-help epitopes be contained in the vaccine, which can be accomplished using added homologous or heterologous T-help epitopes.
METHODE C; peptide syntheseMETHOD C; peptide synthesis
De gewenste B-cal epitopen op de verschillende meningoeoccen klasse 1 buitenmembraaneiwitten worden met behulp van vaste fase peptidesynthese bereid en aan elkaar gekoppeld via daartoe geëigende koppelingsmetho-den, zoals thioether, maleimide, succinimidyl e.a. Er wordt voor gezorgd, dat homologe of heterologe T-help aanwezig zijn. De peptide vaccins worden immunogeen gemaakt via deeltjesvergroting of door verwerking in membraandeeltjes.The desired B-cal epitopes on the various meningococcal class 1 outer membrane proteins are prepared by solid phase peptide synthesis and coupled together by appropriate coupling methods, such as thioether, maleimide, succinimidyl and others. It is ensured that homologous or heterologous T- help be present. The peptide vaccines are made immunogenic via particle enlargement or by incorporation into membrane particles.
TABEL A (zie blz. 4)TABLE A (see page 4)
Bactericide aktiviteit van een kollektie monoclonale antilichamen, gericht tegen het klasse 1 (kl 1), klasse 2/3 (kl 2/3) en lipopolysaccharide (LPS) van meningococcen. (NB = niet bepaald).Bactericidal activity of a collection of monoclonal antibodies, directed against class 1 (cl 1), class 2/3 (cl 2/3) and lipopolysaccharide (LPS) of meningococci. (NB = not determined).
Test stam Bactericide aktiviteit van antistofpool (titer) kl 2/3 pool kl 1 pool LPS poolTest strain Bactericidal activity of antibody pool (titer) kl 2/3 pole kl 1 pole LPS pool
3006 (B:26:P1,2:L2) 1000 8000 NB3006 (W: 26: P1,2: L2) 1000 8000 NB
M981 (B:4:P1 ,*-:L5·) 10 NB 2000M981 (B: 4: P1, * -: L5) 10 NB 2000
M990 ( B: 6: PI. 6: L?) 10 2000 NBM990 (B: 6: PI. 6: L?) 10 2000 NB
M978 (B:8:P1.1:LI,8) NB 8000 1000 M982 (B:9:PI.9:L3,7) 500 2000 1000 H355 (Br 15:PI*15:LI,8) 1000 8000 1000 H44/7 6(B:15:Pi.7.16:L3,7) 1000 8000 4000M978 (B: 8: P1.1: LI, 8) NB 8000 1000 M982 (B: 9: PI.9: L3,7) 500 2000 1000 H355 (Br 15: PI * 15: LI, 8) 1000 8000 1000 H44 / 7 6 (W: 15: Pi. 7.16: L3.7) 1000 8000 4000
Claims (22)
Priority Applications (15)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL8901612A NL8901612A (en) | 1988-12-19 | 1989-06-26 | Vaccines against meningococcal infections - contg. outer membrane proteins or fragments |
| DE68921895T DE68921895T3 (en) | 1988-12-19 | 1989-12-19 | MENINGOCOCCALES CLASS I EXTERNAL MEMBRANE PROTEIN VACCINE. |
| AT90901397T ATE120093T1 (en) | 1988-12-19 | 1989-12-19 | MENINGOCOCCALES CLASS I OUTER MEMBRANE PROTEIN VACCINE. |
| PCT/US1989/005678 WO1990006696A2 (en) | 1988-12-19 | 1989-12-19 | Meningococcal class 1 outer-membrane protein vaccine |
| AU48219/90A AU640118B2 (en) | 1988-12-19 | 1989-12-19 | Meningococcal class 1 outer-membrane protein vaccine |
| EP90901397A EP0449958B9 (en) | 1988-12-19 | 1989-12-19 | Meningococcal class 1 outer-membrane protein vaccine |
| ES90901397T ES2070312T5 (en) | 1988-12-19 | 1989-12-19 | CLASS 1 MENINGOCOCIC EXTERNAL MEMBRANE PROTEIN VACCINE. |
| JP50166290A JP3436756B2 (en) | 1988-12-19 | 1989-12-19 | Meningococcal class I outer membrane protein vaccine |
| CA 2007248 CA2007248C (en) | 1989-01-06 | 1990-01-05 | Meningococcal class 1 outer-membrane protein vaccine |
| PT9280790A PT92807B (en) | 1989-01-06 | 1990-01-08 | PROCESS FOR THE PREPARATION OF AN EXTERNAL MEMBRANE PROTEIN VACCINE MENINGOCOCIA CLASS 1 |
| FI912965A FI113009B (en) | 1988-12-19 | 1991-06-18 | Procedure for Preparing a Vaccine against the Neisseria meningitidis Bacteria and Neisseria meningitidis Strain |
| DK199101174A DK175788B1 (en) | 1988-12-19 | 1991-06-18 | Vaccine against meningococcal diseases, including components thereof, their use and recombinants expressing them |
| NO912369A NO305463B1 (en) | 1988-12-19 | 1991-06-18 | Method of Preparing a Vaccine against Neisseria meningitidis, class 2/3 minus mutant of Neisseria Neisseria meningitidis strain, as well as microorganism expressing a fusion protein |
| US08/204,808 US7118757B1 (en) | 1988-12-19 | 1994-02-15 | Meningococcal class 1 outer-membrane protein vaccine |
| US08/461,651 US7238345B1 (en) | 1988-12-19 | 1995-06-05 | Recombinant microorganism expressing meningococcal class 1 outer membrane protein |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL8803111A NL8803111A (en) | 1988-12-19 | 1988-12-19 | Meningococcus class 1 outer-membrane protein vaccine - useful to immunise against meningococcal disease |
| NL8803111 | 1988-12-19 | ||
| NL8901612A NL8901612A (en) | 1988-12-19 | 1989-06-26 | Vaccines against meningococcal infections - contg. outer membrane proteins or fragments |
| NL8901612 | 1989-06-26 |
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| NL8901612A true NL8901612A (en) | 1990-07-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NL8803111A NL8803111A (en) | 1988-12-19 | 1988-12-19 | Meningococcus class 1 outer-membrane protein vaccine - useful to immunise against meningococcal disease |
| NL8900036A NL8900036A (en) | 1988-12-19 | 1989-01-06 | Meningococcus class 1 outer-membrane protein vaccine - useful to immunise against meningococcal disease |
| NL8901612A NL8901612A (en) | 1988-12-19 | 1989-06-26 | Vaccines against meningococcal infections - contg. outer membrane proteins or fragments |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NL8803111A NL8803111A (en) | 1988-12-19 | 1988-12-19 | Meningococcus class 1 outer-membrane protein vaccine - useful to immunise against meningococcal disease |
| NL8900036A NL8900036A (en) | 1988-12-19 | 1989-01-06 | Meningococcus class 1 outer-membrane protein vaccine - useful to immunise against meningococcal disease |
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| NL (3) | NL8803111A (en) |
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|---|---|---|---|---|
| WO2000071725A2 (en) * | 1999-05-19 | 2000-11-30 | Chiron S.P.A. | Combination neisserial compositions |
| US7604810B2 (en) | 1999-04-30 | 2009-10-20 | Novartis Vaccines And Diagnostics Srl | Conserved Neisserial antigens |
| US8114960B2 (en) | 2000-02-28 | 2012-02-14 | Novartis Ag | Heterologous expression of Neisserial proteins |
| CN101116744B (en) * | 1999-05-19 | 2012-03-07 | 启龙股份公司 | Combination neisserial compositions |
| US8663656B2 (en) | 2002-10-11 | 2014-03-04 | Novartis Ag | Polypeptide-vaccines for broad protection against hypervirulent meningococcal lineages |
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| US10179167B2 (en) | 2010-09-10 | 2019-01-15 | Glaxosmithkline Biologicals S.A. | Developments in meningococcal outer membrane vesicles |
| US10195264B2 (en) | 2004-04-22 | 2019-02-05 | Glaxosmithkline Biologicals S.A. | Immunising against meningococcal serogroup Y using proteins |
| US10376573B2 (en) | 2012-06-14 | 2019-08-13 | Glaxosmithkline Biologicals Sa | Vaccines for serogroup X meningococcus |
-
1988
- 1988-12-19 NL NL8803111A patent/NL8803111A/en not_active Application Discontinuation
-
1989
- 1989-01-06 NL NL8900036A patent/NL8900036A/en not_active Application Discontinuation
- 1989-06-26 NL NL8901612A patent/NL8901612A/en not_active Application Discontinuation
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| US9139621B2 (en) | 1998-05-01 | 2015-09-22 | Glaxosmithkline Biologicals Sa | Neisseria meningitidis antigens and compositions |
| US7604810B2 (en) | 1999-04-30 | 2009-10-20 | Novartis Vaccines And Diagnostics Srl | Conserved Neisserial antigens |
| US9169301B2 (en) | 1999-04-30 | 2015-10-27 | Glaxosmithkline Biologicals Sa | Conserved Neisserial antigens |
| US7862827B2 (en) | 1999-05-19 | 2011-01-04 | Novartis Vaccines And Diagnostics Srl | Combination neisserial compositions |
| WO2000071725A2 (en) * | 1999-05-19 | 2000-11-30 | Chiron S.P.A. | Combination neisserial compositions |
| WO2000071725A3 (en) * | 1999-05-19 | 2001-06-28 | Chiron Spa | Combination neisserial compositions |
| AU783894B2 (en) * | 1999-05-19 | 2005-12-22 | Novartis Vaccines And Diagnostics S.R.L. | Combination neisserial compositions |
| CN101116744B (en) * | 1999-05-19 | 2012-03-07 | 启龙股份公司 | Combination neisserial compositions |
| AU2009200865B2 (en) * | 1999-05-19 | 2012-11-08 | Glaxosmithkline Biologicals S.A. | Combination Neisserial compositions |
| CN100338218C (en) * | 1999-05-19 | 2007-09-19 | 启龙股份公司 | Combination neisserial compositions |
| US8114960B2 (en) | 2000-02-28 | 2012-02-14 | Novartis Ag | Heterologous expression of Neisserial proteins |
| US8703914B2 (en) | 2000-02-28 | 2014-04-22 | Novartis Ag | Heterologous expression of neisserial proteins |
| US9267163B2 (en) | 2000-02-28 | 2016-02-23 | Glaxosmithkline Biologicals Sa | Hybrid expression of neisserial proteins |
| US9011869B2 (en) | 2001-09-06 | 2015-04-21 | Glaxosmithkline Biologicals Sa | Hybrid and tandem expression of Neisserial proteins |
| US8663656B2 (en) | 2002-10-11 | 2014-03-04 | Novartis Ag | Polypeptide-vaccines for broad protection against hypervirulent meningococcal lineages |
| US10195264B2 (en) | 2004-04-22 | 2019-02-05 | Glaxosmithkline Biologicals S.A. | Immunising against meningococcal serogroup Y using proteins |
| US10179167B2 (en) | 2010-09-10 | 2019-01-15 | Glaxosmithkline Biologicals S.A. | Developments in meningococcal outer membrane vesicles |
| US10376573B2 (en) | 2012-06-14 | 2019-08-13 | Glaxosmithkline Biologicals Sa | Vaccines for serogroup X meningococcus |
Also Published As
| Publication number | Publication date |
|---|---|
| NL8900036A (en) | 1990-07-16 |
| NL8803111A (en) | 1990-07-16 |
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