WO2002038174A2 - Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation - Google Patents

Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation Download PDF

Info

Publication number
WO2002038174A2
WO2002038174A2 PCT/DE2001/004213 DE0104213W WO0238174A2 WO 2002038174 A2 WO2002038174 A2 WO 2002038174A2 DE 0104213 W DE0104213 W DE 0104213W WO 0238174 A2 WO0238174 A2 WO 0238174A2
Authority
WO
WIPO (PCT)
Prior art keywords
hantavirus
proteins
vaccines
recombinant
yeast
Prior art date
Application number
PCT/DE2001/004213
Other languages
German (de)
English (en)
Other versions
WO2002038174A3 (fr
Inventor
Rainer Ulrich
Ausra Dargeviciute
Kestutis Sasnauskas
Åke LUNDKVIST
Original Assignee
Humboldt-Universität Zu Berlin Universitätsklinikum Charite
Institute Of Biotechnology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Humboldt-Universität Zu Berlin Universitätsklinikum Charite, Institute Of Biotechnology filed Critical Humboldt-Universität Zu Berlin Universitätsklinikum Charite
Priority to AU2002221553A priority Critical patent/AU2002221553A1/en
Publication of WO2002038174A2 publication Critical patent/WO2002038174A2/fr
Publication of WO2002038174A3 publication Critical patent/WO2002038174A3/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12111—Hantavirus, e.g. Hantaan virus
    • C12N2760/12122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Vaccines containing recombinant hantavirus proteins processes for their preparation and their use
  • the invention relates to vaccines against hantaviruses, processes for their preparation and their use. Areas of application are medicine and veterinary medicine.
  • the invention is intended to enable the highly efficient production of non-infectious, endotoxin-free recombinant hantavirus proteins which are suitable for human use as a vaccine.
  • Hantaviruses are counted among the "emerging viruses" because of their increasing importance as pathogens. They are the causative agent of hemorrhagic fever with renal syndrome, HFRS, (Schmaljohn et al., 1985, Science 227, 1041-1044), which occurs in Europe and Asia, and Hantaviral Pulmonalen, which appeared in America in the early 1990s and was associated with high mortality Syndrome, HPS (Nichol et al., 1993, Science 262, 914-917).
  • HFRS The clinical picture summarized as HFRS (list of abbreviations behind the examples) is caused by different serotypes: infections with the serotypes Hantaan (HTNV) and Dobrava (DOBV) cause the most severe disease, while infections with the serotypes Seoul (SEOV) and Puumala (PUUV) milder show clinical courses.
  • HTNV Hantaan
  • DOBV Dobrava
  • SEOV Dobrava
  • PUUV Puumala
  • RNA genome of negative polarity is composed of 3 segments that encode the viral RNA polymerase, the glycoproteins Gl and G2 and the nucleocapsid protein (N). Because of the endemic spread of hantaviruses in Asia, various inactivated hantavirus vaccines have been produced and tested in China, South and North Korea as well as in Japan (overview in Schmaljohn, 1994, Rev. Med. Virol. 4, 185-196).
  • VACV vaccinia virus
  • a recombinant HTNV-VACV vaccine showed the immunogenicity and safety of the vaccine in a phase I clinical study.
  • a phase II study showed the induction of virus-neutralizing antibodies in% of VACV-naive volunteers, but only about 1 ⁇ of the VACV-immune volunteers (McClain et al., 2000, J. Med. Virol. 60 , 77-85).
  • Sindbis virus-derived constructs were tested, which, however, showed significantly less protection than DNA vaccine constructs (Kamrud et al., 1999).
  • recombinant proteins offer clear advantages in terms of safety (no infectivity, no integration).
  • Heterologous expression systems such as E. coli and insect cells, were able to produce recombinant hantavirus proteins which induced a protective immune response in animal models (Schmaljohn et al., 1990, J. Virol. 64, 3162-3170; Yoshimatsu et al, 1993, Arch Virol.
  • McClain filed patent applications in the USA which relate to hantavirus proteins or to vaccines, in particular to the glycoproteins Gl and G2: US Pat. No. 5,298,423 and 5,614,193.
  • ST Nichol, CFSpiropoulou, TG Ksiazek and PE Rollin protect the detection of the Hantavirus "Four Corners Virus” (US 5945277, WO 9500648), 1995 from this group PE Rollin, L. Elliot, TG Ksiazek and ST Nichol the detection of the "Black Creek Canal Hantavirus” (US 5853980).
  • the invention has for its object to develop new hantavirus vaccines that are safe for human applications.
  • the object was achieved in that with the aid of a yeast expression system which allows the high expression of hantavirus proteins, recombinant hantavirus proteins are isolated in large quantities from yeast cells and purified by simple centrifugation steps and / or by affinity chromatography.
  • the proteins obtained according to the invention have proven to be endotoxin-free and harmless for human applications.
  • the invention relates to the PCR amplification and cloning of hantavirus protein-coding sequences. These DNA sequences are built into yeast expression vectors with a yeast-specific expression unit (promoter and transcription stop signal).
  • the hantavirus proteins are expressed in yeast cells, preferably in Saccharomyces cerevisiae strain fh4c (ATCC # 42368; Colona and Lampen, 1974, Biochemistry 13, 2741-2748). After the yeast cells have been digested with the aid of glass beads, the authentic hantavirus proteins are purified by two successive cesium chloride density centrifugations, hexahistidine-bearing hantavirus proteins by enrichment of the insoluble protein and subsequent nickel chelate chromatography. The subsequent analysis of the recombinant proteins is carried out by SDS-polyacrylamide gel electrophoresis and subsequent silver staining of the gel or immunoblot with hantavirus-specific antibodies.
  • Protectivity of the proteins is provided by immunization of rubella, the natural host of PUUV, and subsequent PUUV exposure (challenge). Protection is provided on the basis of a lack of anti-G2 antibodies in the serum, a lack of N antigen in the lungs and a lack of RNA detection using S-segment-specific PCR.
  • the invention makes a new expression system for hantavirus proteins available which has significant advantages over the previous systems in E. coli or insect cells.
  • yeast expression systems allow simple and inexpensive biotechnological production of antigens.
  • yeasts have no toxins that would cause undesirable side effects in human medical applications.
  • yeast systems are already approved for human vaccine production:
  • the recombinant HBV vaccine is based on yeast-expressed HBsAg. It is particularly surprising that the yeast cells synthesize the hantavirus proteins in large quantities.
  • the recombinant proteins can be isolated and purified in large quantities by simple methods after the lysis of the yeast cells.
  • the proteins according to the invention are endotoxin-free and suitable for human applications as vaccines and in the development of diagnostics.
  • the essence of the invention is the provision of hantavirus vaccines which contain recombinant hantavirus proteins as active components, the proteins in turn being produced in yeast cells.
  • active components they contain nucleocapsid proteins and / or glycoproteins from Hantaviruses, such as the Hantavirus serotypes Puumala, Dobrava and / or Hantaan, in particular the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan-Fojnica.
  • Saccharomyces cerevisiae are used as yeast cells, in particular the fl ⁇ 4c strain.
  • a special feature of the method according to the invention is that a yeast expression plasmid is used which carries a yeast-specific GALIO-PYKI hybrid promoter and the FDHl gene from Candida maltosa as the dominant selection marker.
  • the yeast cells are grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) with 5 mM formaldehyde, after which the synthesis of the hantavirus proteins is induced by adding galactose (final concentration 3%). The yeast cells are disrupted by glass beads.
  • the recombinant hantavirus proteins are purified by two successive cesium chloride density gradient centrifugations or by enrichment of the insoluble proteins and subsequent nickel chelate affinity chromatography.
  • the uses according to the invention of the recombinant hantavirus proteins lie in their use as effective components in hantavirus vaccines and in the development of diagnostics based on the ELISA and / or immunoblot principle.
  • the features of the invention also emerge from the description, the individual features representing advantageous protective designs, individually or in combination in the form of combinations, for which protection is requested with this document.
  • the combination consists of known (hantavirus proteins, centrifugation, affinity chromatography) and new elements (a novel expression system for hantavirus proteins), which mutually influence each other and, in their new overall effect, result in an advantage in use and the desired success, which lies in the fact that now recombinant hantavirus proteins isolated in large quantities from yeast cells, cleaned by simple work steps (centrifugation, chromatography) and made available to the user.
  • hantavirus proteins are used as effective components in hantavirus vaccines.
  • the use of a vaccine made from recombinant hantavirus proteins lies in Induction of protective immunity, for example by immunization with PUUV-His-N protein using aluminum hydroxide as an adjuvant.
  • the invention further consists in the use of a yeast high expression system which allows the production of large amounts of recombinant hantavirus proteins.
  • the recombinant proteins are purified by density gradient centrifugation or affinity chromatography. By extracting the proteins from yeast cells, they are endotoxin-free and suitable for human applications as vaccines and in the development of diagnostics.
  • N-proteins are PCR-amplified according to standard methods using the primers named below carry a J-b ⁇ l restriction site:
  • the PCR products obtained are inserted into the cloning vectors pUC57 (Fermentas, Vilnius, Lithuania), pCRII (Invitrogen, Groningen, the Netherlands) and pCR-Blunt II-TOPO (Invitrogen).
  • the nucleotide sequences of the hantavirus inserts of the plasmids pUC57-VranicaN, pCRII-Slovakia N and pCR-Blunt II-TOPO-Fojnica N are verified by DNA sequencing (FIGS. 1-3).
  • Reverse primer GC TCT AGA ACA TTA AAG CTT AAG CGG CTC CTG
  • the His-PUUV and His-DOBV-PCR amplificates are inserted into the pCR-Blunt II-TOPO vector, the His-HTNV amplificate into the vector pUC57.
  • the complete coding sequence of pCR-Blunt II-TOPO-His-Vranica N is verified by DNA sequencing.
  • only the coding sequences for the N-termini of the N-proteins are determined, and the parts coding for the C-terminus are separated by corresponding fragments from the plasmids pCRII-Slovakia N and pCR-Blunt II-TOPO-Fojnica N. replaced ( Figures 1 to 3).
  • the yeast expression plasmid preferably pFX7 (Scherneck, Sasnauskas and Ulrich, disclosure slip DE 19750220A1, PCT / DE98 / 03329; Sasnauskas et al., 1999, Biol. Chem. 380, 381-386; see FIG. 4), contains a yeast-specific GALIO -PYKI hybrid promoter, - a transcription termination sequence (from PGK1), a 2 ⁇ m DNA fragment,
  • FIG. 5 is as Example of the construction of the pFX7-derived plasmids with PUUV-N coding inserts shown).
  • Recombinant plasmids are selected after transformation in E. coli Kl 2 and characterized by restriction mapping.
  • the pFX7-derived expression plasmids which encode the authentic and His 6 -etailed PUUV, DOBV and HTNV-N proteins are transformed into the yeast Saccharomyces cerevisiae, preferably into the wild-type strain fh4c.
  • 100 ml of YEPD medium 1% yeast extract, 2% peptone, 2%
  • the cell culture is diluted 1: 1 with YEPG induction medium (1% yeast extract, 2% peptone, 6% galactose; with 5 mM formaldehyde) and cultivated for a further 18 hours.
  • the cells are washed with PBS and the cell sediment is frozen at -20 ° C.
  • lysis buffer 0.
  • the yeast cells grown as described under 3. are resuspended after sedimentation in 10 ml of lysis buffer (20 mM PBS, pH 7.6; 2 mM EDTA; 1 mM PMSF) and after adding an equivalent volume of glass beads (diameter 0.5 mm) digested by vortexing for 5 minutes at 4 ° C.
  • lysis buffer 20 mM PBS, pH 7.6; 2 mM EDTA; 1 mM PMSF
  • glass beads diameter 0.5 mm
  • the lysates are centrifuged for 5 minutes at 2000x g.
  • Insoluble proteins are sedimented by centrifugation at 10 OOOxg for 40 minutes (4 ° C). Since the recombinant hantavirus N proteins are found almost exclusively in the sediment, the supernatant is discarded.
  • contaminating cellular proteins are extracted twice by resuspending the pellet in 10 ml extraction buffer (20 mM PBS, pH 7.6; 2 mM EDTA; 1 mM PMSF; 1% Tween 20), shaking for 1 hour at 4 ° C and centrifugation at 10 OOOxg for 30 minutes (4 ° C).
  • the authentic N-proteins in the sediment are then purified by cesium chloride density gradient centrifugation (see 5.), the His 6 -detailed N-derivatives by nickel chelate affinity chromatography (see 6.).
  • the sediment as described under 4. is resuspended in 4 ml extraction buffer.
  • Two ml of the suspension are applied to a preformed cesium chloride gradient (cesium chloride density of 1.1, 1.2, 1.3, 1.4 and 1.5 g / ml in 20 mM PBS, pH 7.6; 2 mM EDTA, 1% Tween 20) layered and centrifuged for 24 hours at 36000 rpm at 4 ° C (Beckman ultracentrifuge).
  • Gradient fractions of 0.5 ml each are characterized by SDS polyacrylamide gel electrophoresis and Western blot analysis. Fractions containing hantavirus protein are subjected to a second cesium chloride density gradient centrifugation.
  • the virus challenge is carried out by inoculation with 10 4 ID o units of the PUUV strain Kazan (Lundkvist et al, 1997, J. Virol. 71, 9515-9523). Three weeks after the challenge, the animals are analyzed for the infection markers. The absence of N-antigen and S-segment-specific RNA in the lungs (determined using PCR; Niklasson and Lundkvist, 1999, In: Manual of Hemorrhagic Fever with renal syndrome and hantavirus pulmonary syndrome; HW Lee, C. Calisher, C.
  • Schmaljohn, Hrg .; WHO Collaborating Center for Virus Reference and Research, Asian Institute for Life Sciences, Seoul) and of G2-specific antibodies in serum (after Challenge) indicates complete sterile protection while the detection of individual infection parameters indicates a partial protection.
  • All 4 immunized animals do not show any of the infection markers, ie they are completely protected from the virus challenge by the immunization (Tab. 1). After challenge, all 3 infection markers are detected in non-vaccinated animals; all 3 infection markers are missing in animals without challenge.
  • rubella mice are immunized with PUUV-His-N protein purified by nickel chelate chromatography using aluminum hydroxide.
  • 3 immunizations with 50 ⁇ g each are administered subcutaneously every 3 weeks.
  • the first two immunizations are carried out with the addition of aluminum hydroxide and the third immunization with the use of PBS.
  • the virus challenge is carried out as described under 7. The missing
  • Table 2 Evidence of the induction of protective immunity by immunization with PUUV-His-N protein using aluminum hydroxide as adjuvant
  • FDH1 gene from Candida maltosa that encodes a formaldehyde dehydrogenase

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des vaccins contre les hanta virus, leur procédé de production et leur utilisation. Ces vaccins ont comme domaine d'application la médecine et la médecine vétérinaire. L'invention concerne également l'utilisation d'un système de forte expression de levure qui permet d'obtenir de grandes quantités de protéines hantavirus recombinantes. Les protéines recombinantes sont nettoyées par centrifugation en gradient de densité ou par chromatographie d'affinité. L'obtention des protéines à partir de cellules de levure garantit qu'elles soient exemptes d'endotoxine et qu'elle conviennent à des applications humaines comme vaccins et dans le développement de diagnostics.
PCT/DE2001/004213 2000-11-08 2001-11-07 Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation WO2002038174A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002221553A AU2002221553A1 (en) 2000-11-08 2001-11-07 Vaccines containing recombinant hantavirus proteins, methods for producing said vaccines and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10055886A DE10055886A1 (de) 2000-11-08 2000-11-08 Impfstoffe, die rekombinante Hantavirusproteine enthalten, Verfahren zu iher Herstellung und ihre Verwendung
DE10055886.0 2000-11-08

Publications (2)

Publication Number Publication Date
WO2002038174A2 true WO2002038174A2 (fr) 2002-05-16
WO2002038174A3 WO2002038174A3 (fr) 2003-08-28

Family

ID=7662912

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2001/004213 WO2002038174A2 (fr) 2000-11-08 2001-11-07 Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation

Country Status (3)

Country Link
AU (1) AU2002221553A1 (fr)
DE (1) DE10055886A1 (fr)
WO (1) WO2002038174A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1916324A2 (fr) 2003-10-27 2008-04-30 Kimberly-Clark Worldwide, Inc. Procédé et appareil pour la production de toiles non tissées
CN101249261B (zh) * 2008-03-18 2010-10-27 中国疾病预防控制中心病毒病预防控制所 一种肾综合征出血热粘膜免疫疫苗及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000648A1 (fr) * 1993-06-24 1995-01-05 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Acides nucleiques d'un nouvel hantavirus et reactifs associes de detection et prevention d'infections
WO1999001153A1 (fr) * 1997-07-03 1999-01-14 The University Of New Mexico Hantavirus du rio mamore
DE19750220A1 (de) * 1997-11-13 1999-05-20 Max Delbrueck Centrum Verfahren zur Gewinnung von virusähnlichen Partikeln auf der Basis von Polyomaviren
WO2000044406A2 (fr) * 1999-01-29 2000-08-03 U.S. Medical Research Institute Of Infectious Diseases Vaccins adn diriges contre les infections a hantavirus

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5916754A (en) * 1995-02-17 1999-06-29 The United States Of America As Represented By The Department Of Health And Human Services Bayou hantavirus and related methods
US5879968A (en) * 1996-11-18 1999-03-09 International Rectifier Corporation Process for manufacture of a P-channel MOS gated device with base implant through the contact window

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000648A1 (fr) * 1993-06-24 1995-01-05 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Acides nucleiques d'un nouvel hantavirus et reactifs associes de detection et prevention d'infections
WO1999001153A1 (fr) * 1997-07-03 1999-01-14 The University Of New Mexico Hantavirus du rio mamore
DE19750220A1 (de) * 1997-11-13 1999-05-20 Max Delbrueck Centrum Verfahren zur Gewinnung von virusähnlichen Partikeln auf der Basis von Polyomaviren
WO2000044406A2 (fr) * 1999-01-29 2000-08-03 U.S. Medical Research Institute Of Infectious Diseases Vaccins adn diriges contre les infections a hantavirus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DARGEVICIUTE A ET AL: "Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, Bd. 20, Nr. 29-30, 4. Oktober 2002 (2002-10-04), Seiten 3523-3531, XP004381816 ISSN: 0264-410X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1916324A2 (fr) 2003-10-27 2008-04-30 Kimberly-Clark Worldwide, Inc. Procédé et appareil pour la production de toiles non tissées
CN101249261B (zh) * 2008-03-18 2010-10-27 中国疾病预防控制中心病毒病预防控制所 一种肾综合征出血热粘膜免疫疫苗及其制备方法

Also Published As

Publication number Publication date
DE10055886A1 (de) 2002-05-29
AU2002221553A1 (en) 2002-05-21
WO2002038174A3 (fr) 2003-08-28

Similar Documents

Publication Publication Date Title
DE69736185T2 (de) Zusammensetzung eines polynucleotid-impfstoffes für katzen
Ahlawat et al. Association of a badnavirus with citrus mosaic disease in India
DE69731309T3 (de) Impfstofformel auf polynukleotidbasis zur behandlung von hundekrankheiten, insbesondere solcher des atmungs- und verdauungstraktes
Boscia et al. Properties of a filamentous virus isolated from grapevines affected by corky bark
DE69532369T2 (de) Rekombinantes infektiöses nicht-in-Segmente-geteiltes, negativ-Strange-RNS-Virus
EP2102345B1 (fr) Proteine f du virus respiratoire syncytial (rsv), et son utilisation
CH679933A5 (fr)
DE69034075T2 (de) Rekombinantes poxvirus und streptokokken enthaltend ein m-protein
DE69935136T2 (de) DNS Pferdeimpfstoffe adjuviert mit Acryl- oder Methacrylpolymersäuren oder EMA (R)
DE69534993T2 (de) Impfstoff aus abgeschwächten hundeparvoviren
DE69929544T2 (de) Attenuierte Pestiviren
DE3137300A1 (de) Zur expression von proteinen von maul- und klauen-seuchen-viren geeignetes dna-molekuel, verfahren zu seiner herstellung und seine verwendung zur erzeugung von proteinen und impfstoffen
DE10143813A1 (de) Infektiöser Rindervirusdiarrhoe-Virusklon
DE69034218T2 (de) Impfstoff und Diagnostikum für den Schweine-Cholera-Virus
WO2002038174A2 (fr) Vaccins contenant des proteines hantavirus recombinantes, leur procede de production et leur utilisation
DE4435907C2 (de) Papillomavirusähnliche Partikel und deren Anwendung
EP0284791B1 (fr) Molécules d'ADN et d'ARN du virus de la méningoencéphalite du printemps précoce sous-type de l'ouest, polypeptides codés par celles-ci ainsi que leur utilisation
DE60208854T2 (de) Attenuierte rekombinante stabile Tollwutvirusmutanten und Lebendimpfstoffe
EP1362125B8 (fr) Procede pour identifier des structures biologiquement actives d'agents pathogenes microbiens
DE2500785C3 (fr)
DE2829089A1 (de) Subunit-vakzine
DE19627193B4 (de) Rekombinantes myxomatisches Virus
DE60035680T2 (de) Komplette gensequenz des esel-leukozyten-impfstoffstammes des equinen infektiösen anämievirus und ihre anwendung
DE60028365T2 (de) Chimäre nukleinsäuren und polypeptide aus lyssavirus
CA2399846A1 (fr) Mutants ehv a gm negative

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WD Withdrawal of designations after international publication

Free format text: DE

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP