WO2002038174A2 - Vaccines containing recombinant hantavirus proteins, methods for producing said vaccines and their use - Google Patents

Vaccines containing recombinant hantavirus proteins, methods for producing said vaccines and their use Download PDF

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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
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hantavirus
proteins
vaccines
recombinant
yeast
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PCT/DE2001/004213
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German (de)
French (fr)
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WO2002038174A3 (en
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Rainer Ulrich
Ausra Dargeviciute
Kestutis Sasnauskas
Åke LUNDKVIST
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Humboldt-Universität Zu Berlin Universitätsklinikum Charite
Institute Of Biotechnology
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Publication of WO2002038174A3 publication Critical patent/WO2002038174A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/12011Bunyaviridae
    • C12N2760/12111Hantavirus, e.g. Hantaan virus
    • C12N2760/12122New 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

Abstract

The invention relates to vaccines against hantaviruses, to methods for producing said vaccines and to their use. The scope of application is medicine or veterinary medicine. The invention is characterised by the use of a yeast high-expression system, which enables large quantities of recombinant hantavirus proteins to be obtained. The recombinant proteins are purified by density gradient centrifugation or affinity chromatography. Obtaining the proteins from yeast cells ensures that they are free of endotoxins and suitable for human applications as vaccines and in the development of diagnostics.

Description

Impfstoffe, die rekombinante Hantavirusproteine enthalten, Verfahren zu ihrer Herstellung und ihre Verwendung Vaccines containing recombinant hantavirus proteins, processes for their preparation and their use
Beschreibungdescription
Die Erfindung betrifft Impfstoffe gegen Hantaviren, Verfahren zu ihrer Herstellung und ihre Verwendung. Anwendungsgebiete sind die Medizin bzw. Veterinärmedizin. Die Erfindung soll die hocheffiziente Produktion von nicht-infektiösen, Endotoxin-freien rekombinanten Hantavirus-Proteinen ermöglichen, die für eine humane Anwendung als Impfstoff geeignet sind.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.
Hantaviren werden wegen ihrer zunehmenden Bedeutung als Krankheitserreger zu den "emerging viruses" gezählt. Sie sind als Erreger des in Europa und Asien auftretenden Hämorrhagischen Fiebers mit Renalem Syndrom, HFRS, (Schmaljohn et al., 1985, Science 227, 1041-1044) und des in Amerika Anfang der 90er Jahre neu aufgetretenen, mit hoher Letalität einhergehenden Hantaviralen Pulmonalen Syndroms, HPS (Nichol et al., 1993, Science 262, 914-917), identifiziert worden. Das als HFRS (Abkürzungsverzeichnis hinter den Ausfuhrungsbeispielen) zusammengefasste Krankheitsbild wird von unterschiedlichen Serotypen hervorgerufen: Den schwersten Krankheitsverlauf verursachen Infektionen mit den Serotypen Hantaan (HTNV) und Dobrava (DOBV), während Infektionen mit den Serotypen Seoul (SEOV) und Puumala (PUUV) mildere klinische Verläufe zeigen. Insgesamt treten weltweit etwa 200000 HFRS-Fälle pro Jahr auf (Krüger und Zöller, 1996, in: Virusdiagnostik, Porstmann, T., Hrg., Blackwell Wissenschaftsverlag, Berlin Wien Oxford, S. 117-128.). In Europa konnte die Ko-Existenz der humanpathogenen Serotypen DOBV und PUUV gezeigt werden (Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411; Sibold et al., 2001, J. Med. Virol., im Druck).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). 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. A total of around 200,000 HFRS cases occur worldwide each year (Krüger and Zöller, 1996, in: Virusdiagnostik, Porstmann, T., ed., Blackwell Wissenschaftsverlag, Berlin Vienna Oxford, pp. 117-128.). In Europe, the coexistence of the human pathogenic serotypes DOBV and PUUV could be shown (Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411; Sibold et al., 2001, J. Med. Virol., In press).
Hantaviren bilden einen separaten Genus innerhalb der Familie B nyaviridae (Schmaljohn et al., 1995, Arch. Virol. Suppl. 10, 300-315). Das RNA-Genom negativer Polarität setzt sich aus 3 Segmenten zusammen, die die virale RNA-Polymerase, die Glykoproteine Gl und G2 sowie das Nukleokapsidprotein (N) kodieren. Wegen der endemischen Verbreitung von Hantaviren in Asien wurden in China, Süd- und Nordkorea sowie in Japan verschiedene inaktivierte Hantavirus-Vakzinen hergestellt und erprobt (Übersicht in Schmaljohn, 1994, Rev. Med. Virol. 4, 185-196). Bisher ist lediglich ein HTNV-Totimpfstoff (Handelsname: Hantavax) in Korea für die humane Anwendung zugelassen worden (Cho and Howard, 1999, Vaccine 17, 2569-2575). Obwohl mit den bisher entwickelten Vollvirus-Totvakzinen z. T. hoffnungsvolle Impferfolge erzielt werden konnten, bleiben eine Reihe von offenen Problemen bestehen: Wegen des hohen Gefährdungspotentials der Hantaviren muss ihre Vermehrung unter L3 -Laborbedingungen erfolgen. Die Virusausbeute bei Kultivierung in Zellkulturen ist sehr gering. Die bei den bisher getesteten Vakzinen beobachtete geringe Virus-neutralisierende Aktivität wird möglicherweise durch den nur geringen Anteil von Gl und G2 am Gesamtprotein sowie die Zerstörung der Gl- und G2-Konformation bei der Virusinaktivierung verursacht. Eine Lösungsmöglichkeit für die genannten Probleme ist die Herstellung von rekombinanten Virus-Untereinheiten- Vakzinen. In Tierexperimenten konnte durch Immunisierung mit Vaccinia Virus (VACV)-abgeleiteten Vakzinen, die die kompletten M-Segmente von HTNV bzw. SEOV trugen, eine schützende Immunantwort gegen das entsprechende homologe Virus (Schmaljohn et al., 1990, J. Virol. 64, 3162-3170; Xu et al, 1992, Am. J. Trop. Med. Hyg. 47, 397-404) induziert werden. Während bei Immunisierung mit HTNV- und SEOV-VACV- Rekombinanten auch gegen SEOV und HTNV geschützt wurde, wurde gegen einen PUUV- Challenge nicht geschützt (Chu et al, 1995, J. Virol. 69, 6417-6423).Hantaviruses form a separate genus within the B nyaviridae family (Schmaljohn et al., 1995, Arch. Virol. Suppl. 10, 300-315). The 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). So far, only one HTNV vaccine (trade name: Hantavax) has been used in Korea for human use been approved (Cho and Howard, 1999, Vaccine 17, 2569-2575). Although with the previously developed full virus dead vaccines z. Hopeful vaccination successes have been achieved, a number of open problems remain: Because of the high risk potential of hantaviruses, their multiplication must take place under L3 laboratory conditions. The virus yield when cultivated in cell cultures is very low. The low virus-neutralizing activity observed in the vaccines tested so far may be caused by the low proportion of Gl and G2 in the total protein and the destruction of the Gl and G2 conformation during virus inactivation. One solution to the problems mentioned is the production of recombinant virus subunit vaccines. In animal experiments, a protective immune response against the corresponding homologous virus was able to be obtained by immunization with vaccinia virus (VACV)-derived vaccines which carried the complete M segments of HTNV or SEOV (Schmaljohn et al., 1990, J. Virol. 64, 3162-3170; Xu et al, 1992, Am. J. Trop. Med. Hyg. 47, 397-404). While immunization with HTNV and SEOV-VACV recombinants also protected against SEOV and HTNV, protection against a PUUV challenge was not provided (Chu et al, 1995, J. Virol. 69, 6417-6423).
Eine rekombinante HTNV- VACV- Vakzine zeigte in einer klinischen Phase I- Studie die Immunogenität und Sicherheit der Vakzine. Eine Phase Il-Studie zeigte bei Immunisierung zwar bei % von VACV-naiven Probanden die Induktion von Virus-neutralisierenden Antikörpern, jedoch nur etwa bei lλ der VACV-immunen Probanden (McClain et al., 2000, J. Med. Virol. 60, 77-85).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 λ of the VACV-immune volunteers (McClain et al., 2000, J. Med. Virol. 60 , 77-85).
Die bisher hergestellten nackten DNA- Vakzinen auf der Basis von Gl/G2-kodierenden Konstrukten zeigten in Tiermodellen die Induktion virus-neutralisierender Antikörper und Protektion an (Hooper et al, 1999, Virology 255, 269-278; Bharadwaj et al, 1999, Vaccine 17, 2836-2843; Kamrud et al., 1999, Virology 263, 209-219). Dagegen konnte bei Inokulation von N-kodierenden Konstrukten keine bzw. nur eine schwache Protektion induziert werden. Für eine klinische Anwendung von DNA-Vakzinen sind jedoch noch eine Reihe von Fragen bezüglich der Sicherheit (Gefahr der Toleranzinduktion, Integration der DNA, Autoimmunität) zu untersuchen (Davis and Cluskie, 1999, Microbes and Infection 1, 7-21). Alternativ wurden Sindbisvirus-abgeleitete Konstrukte getestet, die jedoch eine deutlich geringere Protektion als DNA-Vakzine-Konstrukte zeigten (Kamrud et al., 1999). Gegenüber den bisher genannten Systemen bieten rekombinante Proteine bezüglich der Sicherheit deutliche Vorteile (keine Infektiosität, keine Integration). In einer Reihe von heterologen Expressionssystemen, wie E. coli und Insektenzellen, konnten rekombinante Hantavirus-Proteine hergestellt werden, die in Tiermodellen eine protektive Immunantwort induzierten (Schmaljohn et al., 1990, J. Virol. 64, 3162-3170; Yoshimatsu et al, 1993, Arch. Virol. 130, 365-376; Lundkvist et al., 1996, Virology 216, 397-406; Ulrich et al., 1998, Vaccine 16, 272-280). Während E. co/z'-Expressionssysteme eine Hochexpression von Hantavirus-Proteinen erlauben, stellt eine mögliche Kontamination mit bakteriellen Endotoxinen ein Problem für die humane Anwendung dar. Demgegenüber bieten Insektenzell-Expressionssysteme den Vorteil, frei von Endotoxinen zu sein, sind jedoch in ihrer Syntheseleistung für Fremdproteine den E. coli- Systemen unterlegen. Die Geschichte der Entdeckung und der Bekämpfung von Hantaviren lässt sich auch anhand der Patentliteratur dokumentieren. C.S. Schmaljohn und J. Dalrymple hatten zunächst 1987 und dann 1991 zusammen mit DJ. McClain in den USA Patentanmeldungen hinterlegt, die sich auf Hantavirus-Proteine bzw. auf Vakzine beziehen, insbesondere auf die Glykoproteine Gl und G2: US-PS (PS = Patentschrift) 5298423 und 5614193. 1993 ließen sich S.T. Nichol, C.F.Spiropoulou, T.G. Ksiazek und P.E. Rollin den Nachweis des Hantavirus "Four Corners Virus" schützen (US 5945277, WO 9500648), 1995 aus dieser Gruppe P.E. Rollin, L. Elliot, T.G. Ksiazek und S.T. Nichol den Nachweis des "Black Creek Canal Hantavirus" (US 5853980). In Korea meldeten 1996 H.S. Kim, W.D. Yoo, S.O. Kim, K.S. Noh und S.P. Hong "Hantaan virus nucleocapsid protein expressed from E. coli" (WO 9727302) zum Patent an, im gleichen Jahr in Schweden (SE) Ä. Lundkvist einen Impfstoff gegen Hantavirus oder Puumalavirus auf der Basis des Nukleokapsid-Proteins (N) (WO 9728819). In den USA folgten 1997 B.L. Hjelle und N. Torrez-Martinez mit einer Nukleokapsidprotein- Vakzine des "Rio Mamore Hantavirus" (WO 9901153), 1998 in Korea H.W. Lee, S.J. Ryu, CN. Ahn, H. Kim und T. Tomiyama mit einer Anmeldung des Titels "Diagnostic reagent for Puumala virus infection" (EP 952450). Auf das Jahr 1999 gehen die USA- Anmeldungen von S.T. Nichol, S. Morzunov, T.G. Ksiazek und P.E. Rollin über die Entdeckung und Isolierung des neuen Hantavirus "Bayou" (US 5916754) sowie von C.S. Schmaljohn und J.W. Hooper über eine DNA- Vakzine gegen Hantavirus-Infektionen (WO 2000044406) zurück. Die aufgezeigten Lösungen lassen jedoch die bereits oben erwähnten Probleme von rekombinanten Proteinen aus E. coli, von DNA- Vakzinen und VACV-abgeleiteten Lebendvakzinen offen.The previously produced naked DNA vaccines based on Gl / G2-coding constructs indicated the induction of virus-neutralizing antibodies and protection in animal models (Hooper et al, 1999, Virology 255, 269-278; Bharadwaj et al, 1999, Vaccine 17, 2836-2843; Kamrud et al., 1999, Virology 263, 209-219). In contrast, no or only a weak protection could be induced when inoculating N-coding constructs. For a clinical application of DNA vaccines, however, a number of questions regarding safety (risk of tolerance induction, integration of DNA, autoimmunity) have to be examined (Davis and Cluskie, 1999, Microbes and Infection 1, 7-21). Alternatively, Sindbis virus-derived constructs were tested, which, however, showed significantly less protection than DNA vaccine constructs (Kamrud et al., 1999). Compared to the previously mentioned systems, recombinant proteins offer clear advantages in terms of safety (no infectivity, no integration). In a series of 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. 130, 365-376; Lundkvist et al., 1996, Virology 216, 397-406; Ulrich et al., 1998, Vaccine 16, 272-280). While E. co / z ' expression systems allow high expression of hantavirus proteins, possible contamination with bacterial endotoxins is a problem for human use. In contrast, insect cell expression systems offer the advantage of being free of endotoxins, but are in theirs Inferior synthesis performance for foreign proteins to the E. coli systems. The history of the discovery and control of hantaviruses can also be documented using the patent literature. CS Schmaljohn and J. Dalrymple first had 1987 and then 1991 together with DJ. 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. In 1993, 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). In 1996, HS Kim, WD Yoo, SO Kim, KS Noh and SP Hong applied for a patent for "Hantaan virus nucleocapsid protein expressed from E. coli" (WO 9727302), in the same year in Sweden (SE) Ä. Lundkvist is a vaccine against Hantavirus or Puumala virus based on the nucleocapsid protein (N) (WO 9728819). In the USA in 1997 BL Hjelle and N. Torrez-Martinez followed with a nucleocapsid protein vaccine from the "Rio Mamore Hantavirus" (WO 9901153), in 1998 in Korea HW Lee, SJ Ryu, CN. Ahn, H. Kim and T. Tomiyama with an application for the title "Diagnostic reagent for Puumala virus infection" (EP 952450). In 1999, the US applications by ST Nichol, S. Morzunov, TG Ksiazek and PE Rollin for the discovery and isolation of the new Hanta virus "Bayou" (US 5916754), as well as CS Schmaljohn and JW Hooper for a DNA vaccine Hantavirus infections (WO 2000044406). However, the solutions shown leave open the problems already mentioned above of recombinant proteins from E. coli, of DNA vaccines and VACV-derived live vaccines.
Der Erfindung liegt die Aufgabe zugrunde, neue Hantavirus-Impfstoffe zu entwickeln, die für humane Anwendungen unbedenklich sind. Die Aufgabe wurde dadurch gelöst, dass mit Hilfe eines Hefe-Expressionssystems, das die Hochexpression von Hantavirus-Proteinen erlaubt, rekombinante Hantavirus-Proteine in großer Menge aus Hefezellen isoliert und durch einfache Zentrifugationsschritte und/oder durch Affmitätschromatografie gereinigt werden. Die erfindungsgemäß gewonnenen Proteine haben sich als Endotoxin-frei und für humane Anwendungen unbedenklich erwiesen. Die Erfindung hat die PCR-Amplifikation und Klonierung von Hantavirusprotein- kodierenden Sequenzen zum Inhalt. Diese DNA-Sequenzen werden in Hefeexpressionsvektoren mit einer Hefe-spezifischen Expressionseinheit (Promoter und Transkriptionsstopsignal) eingebaut. Die Expression der Hantavirusproteine erfolgt in Hefezellen, bevorzugt in Saccharomyces cerevisiae Stamm fh4c (ATCC #42368; Colona und Lampen, 1974, Biochemistry 13, 2741-2748). Nach Aufschluss der Hefezellen mit Hilfe von Glasperlen werden die authentischen Hantavirusproteine durch zwei aufeinanderfolgende Cäsiumchlorid-Dichtezentrifugationen, Hexahistidin-tragende Hantavirusproteine durch Anreicherung des unlöslichen Proteins und anschließende Nickelchelatchromatografie gereinigt. Die anschließende Analyse der rekombinanten Proteine erfolgt durch SDS- Polyacrylamidgelelektrophorese und anschließende Silberfärbung des Gels bzw. Immunoblot mit Hantavirus-spezifischen Antikörpern. Die Protektivität der Proteine wird durch Immunisierung von Rötelmäusen, dem natürlichen Wirt des PUUV, und anschließende PUUV-Belastung (Challenge) erbracht. Der Schutz wird anhand von fehlenden anti-G2- Antikörpern im Serum, fehlenden N-Antigens in der Lunge und fehlenden RNA-Nachweises mittels S-Segment-spezifischer PCR erbracht.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.
Durch die Erfindung wird ein neuartiges Expressionssystem für Hantavirusproteine verfügbar, das gegenüber den bisherigen Systemen in E. coli bzw. Insektenzellen wesentliche Vorteile aufweist. So erlauben Hefe-Expressionssysteme eine einfache und kostengünstige biotechnologische Produktion von Antigenen. Des weiteren besitzen Hefen keine Toxine, die unerwünschte Nebenwirkungen bei humanmedizinischen Anwendungen hervorrufen würden. Ein weiterer Vorteil besteht darin, dass Hefesysteme bereits für die humane Vakzineproduktion zugelassen sind: Die rekombinante HBV- Vakzine basiert auf Hefe- exprimiertem HBsAg. Es ist besonders überraschend, dass die Hefezellen die Hantavirusproteine in großer Menge synthetisieren. Außerdem können die rekombinanten Proteine nach der Lyse der Hefezellen in großer Menge mit einfachen Methoden isoliert und gereinigt werden. Durch die Art der Gewinnung aus Hefezellen sind die erfindungsgemäßen Proteine Endotoxin-frei und für humane Anwendungen als Impfstoffe sowie in der Diagnostikaentwicklung geeignet. Das Wesen der Erfindung besteht in der Bereitstellung von Hantavirus-Impfstoffen, die als wirksame Komponenten rekombinante Hantavirusproteine enthalten, wobei die Proteine ihrerseits in Hefezellen hergestellt werden. Als wirksame Komponenten enthalten sie Nukleokapsidproteine und/oder Glykoproteine von Hantaviren, wie der Hantavirus-Serotypen Puumala, Dobrava und/oder Hantaan, insbesondere der Hantavirus-Stämme Puumala- Vranica/Hällnäs, Dobrava-Slovakia und/oder Hantaan-Fojnica. Als Hefezellen finden Saccharomyces cerevisiae Verwendung, insbesondere des Stammes flι4c. Ein besonderes Merkmal des erfmdungsgemäßen Verfahrens besteht darin, dass ein Hefeexpressionsplasmid eingesetzt wird, das einen Hefe-spezifischen GALIO-PYKI-Hybrid- Promoter und das FDHl-Gen von Candida maltosa als dominanten Selektionsmarker trägt. Die Hefezellen werden in YEPD-Medium (1% Hefe-Extrakt, 2%Pepton, 2% Glukose) mit 5 mM Formaldehyd angezüchtet, danach wird durch Zugabe von Galaktose (Endkonzentration 3%) die Synthese der Hantavirusproteine induziert. Der Aufschluss der Hefezellen erfolgt durch Glasperlen. Die rekombinanten Hantavirusproteine werden durch zwei aufeinanderfolgende Cäsiumchlorid-Dichtegradienten-Zentrifugationen oder durch Anreicherung der unlöslichen Proteine und anschließende Nickelchelat-Affinitätschromatografϊe gereinigt. Die erfmdungsgemäßen Verwendungen der rekombinanten Hantavirusproteine liegen in ihrem Einsatz als wirksame Komponenten in Hantavirus-Impfstoffen sowie in der Diagnostikaentwicklung basierend auf dem ELISA- und/oder Immunoblot-Prinzip.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. For example, yeast expression systems allow simple and inexpensive biotechnological production of antigens. Furthermore, yeasts have no toxins that would cause undesirable side effects in human medical applications. Another advantage is that 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. In addition, the recombinant proteins can be isolated and purified in large quantities by simple methods after the lysis of the yeast cells. Because of the way they are obtained from 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. As 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 flι4c 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.
Die Merkmale der Erfindung gehen außer aus den Ansprüchen auch aus der Beschreibung hervor, wobei die einzelnen Merkmale jeweils für sich allein oder zu mehreren in Form von Kombinationen vorteilhafte schutzfähige Ausführungen darstellen, für die mit dieser Schrift Schutz beantragt wird. Die Kombination besteht aus bekannten (Hantavirusproteinen, Zentrifugation, Affmitätschromatografie) und neuen Elementen (ein neuartiges Expressionssystem für Hantavirusproteine), die sich gegenseitig beeinflussen und in ihrer neuen Gesamtwirkung einen Gebrauchsvorteil und den erstrebten Erfolg ergeben, der darin liegt, dass nunmehr rekombinante Hantavirus-Proteine in großer Menge aus Hefezellen isoliert, durch einfache Arbeitsschritte (Zentrifugation, Chromatografie) gereinigt und dem Anwender zur Verfügung gestellt werden können.In addition to the claims, 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.
Die erfindungsgemäße Verwendung der rekombinanten Hantavirusproteine liegt darin, dass sie als wirksame Komponenten in Hantavirus-Impfstoffen eingesetzt werden. Die Verwendung einer aus rekombinanten Hantavirusproteinen hergestellten Vakzine liegt in der Induktion einer protektiven Immunität, z.B. durch Immunisierung mit PUUV-His-N-Protein unter Verwendung von Aluminiumhydroxid als Adjuvanz.The use of the recombinant hantavirus proteins according to the invention is that they 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.
Die Erfindung besteht weiterhin in der Verwendung eines Hefe-Hochexpressionssystems, das die Gewinnung großer Mengen rekombinanter Hantavirus-Proteine erlaubt. Die rekombinanten Proteine werden durch Dichtegradientenzentrifugation oder Affinitätschromatografie gereinigt. Durch die Gewinnung der Proteine aus Hefezellen sind diese Endotoxin-frei und für humane Anwendungen als Impfstoffe sowie in der Diagnostikaentwicklung geeignet.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.
Die Erfindung soll nachfolgend durch ein Ausführungsbeispiel näher erläutert werden, ohne auf dieses Beispiel beschränkt zu sein.The invention is to be explained in more detail below using an exemplary embodiment, without being restricted to this example.
Ausführungsbeispielembodiment
1. Gewinnung der Hantavirus-kodierenden Sequenzen1. Obtaining the hantavirus coding sequences
Die kompletten offenen Leserahmen für PUUV- (Stamm Vranica/Hällnäs; Reip et al, 1995, Arch. Virol. 140, 2011-2026), DOBV- (Slowakischer Stamm 862; Sibold et al, 2001, J. Med. Virol., im Druck) und HTNV- (Stamm Fojnica; Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411) N-Proteine werden nach Standardverfahren PCR-amplifiziert unter Verwendung der nachfolgend genannten Primer, die einen J-bαl-Restriktionsort tragen:The complete open reading frames for PUUV- (strain Vranica / Hällnäs; Reip et al, 1995, Arch. Virol. 140, 2011-2026), DOBV- (Slovak strain 862; Sibold et al, 2001, J. Med. Virol., in print) and HTNV- (strain Fojnica; Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411) N-proteins are PCR-amplified according to standard methods using the primers named below carry a J-bαl restriction site:
PUUV (Vranica/ Hällnäs; Figur 1) Forward primer: ATA TCT AGA AC A ATG AGT GAT CTG AC A G Reverse primer: TAT TCT AGA TTA TAT CTT AAG TGG ATC CTPUUV (Vranica / Hällnäs; Figure 1) Forward primer: ATA TCT AGA AC A ATG AGT GAT CTG AC A G Reverse primer: TAT TCT AGA TTA TAT CTT AAG TGG ATC CT
DOBV (Slovakia 862; Figur 2) Forward primer:DOBV (Slovakia 862; Figure 2) Forward primer:
GCTCTAGAACAATGGCAACAYTAGAGGAACTCGCTCTAGAACAATGGCAACAYTAGAGGAACTC
Reverse primer:Reverse primer:
GC TCTAGAACATTAAAG CTTAAGCGGCTC YTG HTNV (Fojnica; Figur 3) Forward primer:GC TCTAGAACATTAAAG CTTAAGCGGCTC YTG HTNV (Fojnica; Figure 3) Forward primer:
GC TCT AGA ACA ATG GCA ACT ATG GAG GAA TTA Reverse primer:GC TCT AGA ACA ATG GCA ACT ATG GAG GAA TTA Reverse primer:
GC TCT AGA TTA GAG TTT CAA AGG CTC TTGGC TCT AGA TTA GAG TTT CAA AGG CTC TTG
Die erhaltenen PCR-Produkte werden in die Klonierungsvektoren pUC57 (Fermentas, Vilnius, Litauen), pCRII (Invitrogen, Groningen, Niederlande) bzw. pCR-Blunt II-TOPO (Invitrogen) inseriert. Durch DNA-Sequenzierung werden die Nukleotidsequenzen der Hantavirus-Inserts der Plasmide pUC57-VranicaN, pCRII-Slovakia N und pCR-Blunt II- TOPO-Fojnica N verifiziert (Figuren 1-3).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).
Um 6 Histidin-Kodonen mit dem 5 '-Ende der N-Protein-kodierenden Sequenzen zu fusionieren, werden die Hantavirus-Sequenzen in den Plasmiden pUC57-VranicaN, pCRII- Slovakia N und pCR-Blunt II-TOPO-Fojnica N mit folgenden Primern PCR-amplifiziert (Figuren 1-3):In order to fuse 6 histidine codons with the 5 'end of the N-protein coding sequences, the hantavirus sequences in the plasmids pUC57-VranicaN, pCRII-Slovakia N and pCR-Blunt II-TOPO-Fojnica N with the following primers PCR amplified (Figures 1-3):
His-PUUV (Vranica Hällnäs; Figur 1) Forward primer:His-PUUV (Vranica Hällnäs; Figure 1) Forward primer:
TACT AGTATG CAC CAT CAC CAT CAC CATAGT GAT CTGACA GATTACT AGTATG CAC CAT CAC CAT CAC CATAGT GAT CTGACA GAT
Reverse primer:Reverse primer:
T ACT AGT TTA TAT CTT AAG TGG ATC CTT ACT AGT TTA TAT CTT AAG TGG ATC CT
His-DOBV (Slovakia 862; Figur 2)His-DOBV (Slovakia 862; Figure 2)
Forward primer:Forward primer:
A TCT AGAACAATG CAC CAT CAC CAT CAC CAT GCAACA CTA GAG GAA CTCA TCT AGAACAATG CAC CAT CAC CAT CAC CAT GCAACA CTA GAG GAA CTC
CC
Reverse primer: GC TCT AGA ACA TTA AAG CTT AAG CGG CTC CTGReverse primer: GC TCT AGA ACA TTA AAG CTT AAG CGG CTC CTG
His-HTNV (Fojnica; Figur 3) Forward primer: ATCT AGAACAATG CAC CAT CAC CAT CAC CAT GCAACTATGGAG GAATTAHis-HTNV (Fojnica; Figure 3) Forward primer: ATCT AGAACAATG CAC CAT CAC CAT CAC CAT GCAACTATGGAG GAATTA
CAGCAG
Reverse primer:Reverse primer:
GC TCT AGA TTA GAG TTT CAA AGG CTC TTGGC TCT AGA TTA GAG TTT CAA AGG CTC TTG
Die His-PUUV- und His-DOBV-PCR-Amplifikate werden in den pCR-Blunt II-TOPO- Vektor , das His-HTNV-Amplifikat in den Vektor pUC57 inseriert. Die komplette kodierende Sequenz von pCR-Blunt II-TOPO-His-Vranica N wird durch DNA-Sequenzierung verifiziert. Bei den beiden anderen Plasmiden werden nur die kodierenden Sequenzen für die N-Termini der N-Proteine bestimmt, und die C-Terminus-kodierenden Teile werden durch entsprechende Fragmente aus den Plasmiden pCRII-Slovakia N und pCR-Blunt II-TOPO-Fojnica N ersetzt (Figuren 1 bis 3).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. In the case of the other two plasmids, 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).
Figuren 1 bis 3 PCR-Amplifikation und Klonierung der Hantavirus-N-Protein-kodierenden SequenzenFigures 1 to 3 PCR amplification and cloning of the hantavirus N protein coding sequences
Legende zu den Figuren 1 bis 3 : Figur 1 :Legend for Figures 1 to 3: Figure 1:
B Histidine tail generation A Construction of yeast expression vector for non His tagged N Protein C Construction of yeast expression vector for His tagged N ProteinB Histidine tail generation A Construction of yeast expression vector for non His tagged N Protein C Construction of yeast expression vector for His tagged N Protein
Figuren 2 und 3Figures 2 and 3
A Histidine tail generation B Construction of yeast expression vector for non His tagged N Protein C Construction of yeast expression vector for His tagged N ProteinA Histidine tail generation B Construction of yeast expression vector for non His tagged N Protein C Construction of yeast expression vector for His tagged N Protein
2. Herstellung von Hefe-Expressionsvektoren2. Preparation of yeast expression vectors
Das Hefeexpressionsplasmid, vorzugsweise pFX7 (Scherneck, Sasnauskas und Ulrich, Offenlegungssclirift DE 19750220A1, PCT/DE98/03329; Sasnauskas et al., 1999, Biol. Chem. 380, 381-386; siehe Figur 4), enthält einen Hefe-spezifischen GALIO-PYKI-Hybrid-Promoter, - eine Transkriptionsterminationssequenz (von PGK1), - ein 2 μm DNA-Fragment,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 μm DNA fragment,
- das FDHl-Gen von Candida maltosa als dominanten Selektionsmarker- The FDHl gene from Candida maltosa as the dominant selection marker
- und einen unikalen Restriktionsort für die Restriktionsendonuklease Xbal zur Insertion von Fremdsequenzen, lokalisiert zwischen Promoter und Transkriptionsterminator. Die Hantavirus N-Protein-kodierenden Sequenzen (mit eigenem Translationsinitiations- und - terminationssignal) werden durch Xbal- bzw. S^el-Spaltungen aus den Klonierungsvektoren isoliert und in den mit Xbal linearisierten Hefeexpressionsvektor pFX7 inseriert (in Figur 5, Figur 6 ist als Beispiel der Aufbau der pFX7-abgeleiteten Plasmide mit PUUV-N-kodierenden Inserts gezeigt). Rekombinante Plasmide werden nach Transformation in E. coli Kl 2 selektioniert und durch Restriktionskartierung charakterisiert.- And a unique restriction site for the restriction endonuclease Xbal for the insertion of foreign sequences, located between the promoter and transcription terminator. The Hantavirus N-protein coding sequences (with their own translation initiation and termination signal) are isolated from the cloning vectors by Xbal or S ^ el cleavages and inserted into the ybal expression vector pFX7 linearized with Xbal (in FIG. 5, FIG. 6 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.
Figuren 4 bis 6Figures 4 to 6
Aufbau der pFX7-abgeleiteten Expressionsplasmide Figur 4: pFX7 Figur 5 : pFX7-PUUV-NStructure of the pFX7-derived expression plasmids Figure 4: pFX7 Figure 5: pFX7-PUUV-N
Figur 6: pFX7-His6 -PUUV-NFigure 6: pFX7-His 6 -PUUV-N
3. Expression der Hantavirus-Nukleokapsidproteine in Hefe3. Expression of Hantavirus nucleocapsid proteins in yeast
Die pFX7-abgeleiteten Expressionsplasmide, die die authentischen und His 6-getailten PUUV-, DOBV- und HTNV-N-Proteine kodieren, werden in die Hefe Saccharomyces cerevisiae, vorzugsweise in den Wildtypstamm fh4c, transformiert. Zur Anzucht der transformierten Hefezellen werden 100 ml YEPD-Medium (1% Hefe-Extrakt, 2%Pepton, 2%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. To grow the transformed yeast cells, 100 ml of YEPD medium (1% yeast extract, 2% peptone, 2%
Glukose; mit 5 mM Formaldehyd) mit den Hefezellen beimpft und 24 Stunden bei 30 °C unterglucose; with 5 mM formaldehyde) inoculated with the yeast cells and under for 24 hours at 30 ° C
Schütteln kultiviert. Die Zellkultur wird 1:1 mit YEPG-Induktionsmedium (1% Hefe-Extrakt, 2%Pepton, 6% Galaktose; mit 5 mM Formaldehyd) verdünnt und weitere 18 Stunden kultiviert. Die Zellen werden mit PBS gewaschen und das Zellsediment bei -20 °C eingefroren. Für die Expressionskontrolle wird ein Aliquot entnommen und mit Lysepuffer (0,Shake cultivated. 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. For the expression control, an aliquot is taken and washed with lysis buffer (0,
125 M Tris-HCl, pH 6,8; 4% SDS, 20% Glycerin, 10% 2-Mercaptoethanol, 0,2 mg/10 ml125 M Tris-HCl, pH 6.8; 4% SDS, 20% glycerin, 10% 2-mercaptoethanol, 0.2 mg / 10 ml
Bromphenolblau) denaturiert. Nach Auftrennung im SDS-Polyacrylamidgel werden die rekombinanten Hantavirus-N-Proteine im Western blot unter Verwendung von N- und His6- spezifϊschen Antikörpern nachgewiesen (Figur 7).Bromophenol blue) denatured. After separation in the SDS polyacrylamide gel, the recombinant hantavirus N proteins are detected in a Western blot using N- and His 6 -specific antibodies (FIG. 7).
Figur 7 Nachweis der rekombinanten Hantavirus-N-Proteine in Totallysaten von Hefezellen mittels Western blot unter Verwendung des N- spezifischen Antikörpers 1C12 (A) und eines His6- spezifischen Antikörpers (B)Figure 7 Detection of the recombinant hantavirus N proteins in totaly lysates of yeast cells by means of Western blot using the N-specific antibody 1C12 (A) and a His 6 -specific antibody (B)
1- DOBV-His-N; 2-DOBV-N; 3-HTNV-His-N; 4-HTNV-N; 5-PUUV-His-N; 6- Negativkontrolle (pFX7).1- DOBV-His-N; 2-DOBV N; 3-HTNV-His-N; 4-HTNV N; 5-PUUV-His-N; 6- Negative control (pFX7).
4. Aufschluss der Hefezellen und Anreicherung der Hantavirus-Nukleokapsidproteine4. Disruption of the yeast cells and enrichment of the hantavirus nucleocapsid proteins
Die, wie unter 3. beschrieben, angezüchteten Hefezellen werden nach Sedimentation in 10 ml Lysepuffer (20 mM PBS, pH 7,6; 2mM EDTA; 1 mM PMSF) resuspendiert und nach Zugabe eines äquivalenten Volumens von Glasperlen (Durchmesser 0,5 mm) durch Vortexing für 5 Minuten bei 4 °C aufgeschlossen. Zur Sedimentierung der Zellbruchstücke werden die Lysate 5 Minuten bei 2000x g zentrifugiert. Unlösliche Proteine werden durch Zentrifugation bei 10 OOOxg für 40 Minuten (4 °C) sedimentiert. Da die rekombinanten Hantavirus-N-Proteine fast ausschließlich im Sediment gefunden werden, wird der Überstand verworfen. Um die N- Proteine in der Pelletfraktion weiter anzureichern, werden kontaminierende zelluläre Proteine zwei Mal extrahiert durch Resuspendieren des Pellets in 10 ml Extraktionspuffer (20 mM PBS, pH 7,6; 2mM EDTA; 1 mM PMSF; 1% Tween 20), Schütteln für 1 Stunde bei 4 °C und Zentrifugation bei 10 OOOxg für 30 Minuten (4 °C). Die im Sediment befindlichen authentischen N-Proteine werden anschließend durch Cäsiumchlorid-Dichtegradienten- Zentrifugation (siehe 5.), die His6-getailten N-Derivate durch Nickelchelat- Affmitätschromatografie gereinigt (siehe 6.).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. To sediment the cell fragments, 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. To further enrich the N proteins in the pellet fraction, 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.).
5. Reinigung der authentischen Hantavirus-Nukleokapsidproteine5. Purification of the authentic Hantavirus nucleocapsid proteins
Das Sediment, wie unter 4. beschrieben, wird in 4 ml Extraktionspuffer resuspendiert. Zwei ml der Suspension werden auf einen vorgeformten Cäsiumchloridgradienten (Cäsiumchlorid- Dichte von 1,1, 1,2, 1,3, 1,4 und 1,5 g/ml in 20 mM PBS, pH 7,6; 2 mM EDTA, 1% Tween 20) geschichtet und 24 Stunden bei 36000 U/min bei 4 °C (Beckman-Ultrazentrifuge) zentrifugiert. Gradientenfraktionen von jeweils 0,5 ml werden durch SDS- Polyacrylamidgelelektrophorese und Western blot-Analyse charakterisiert. Hantavirus- Protein-enthaltende Fraktionen werden einer zweiten Cäsiumchloriddichtegradienten- Zentrifugation unterzogen. Positive Gradientenfraktionen werden gegen PBS dialysiert, aliquotiert und bei - 20 °C gelagert. Die Ausbeute der 3 rekombinanten Hantavirusproteine, die nach der Bradfordmethode (Bradford-Reagenz von BioRad, München, Deutschland; Bradford, 1976, Analyt. Biochem. 72, 248-254) bestimmt wurde, liegt im Durchschnitt bei etwa 1,5 mg pro g Feuchtgewicht (entspricht ca. 18 mg pro 1 Kulturvolumen).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. Positive gradient fractions are dialyzed against PBS, aliquoted and stored at -20 ° C. The yield of the 3 recombinant hantavirus proteins, which were prepared using the Bradford method (Bradford reagent from BioRad, Munich, Germany; Bradford, 1976, analyte. Biochem. 72, 248-254) was determined, is on average about 1.5 mg per g wet weight (corresponds to about 18 mg per 1 culture volume).
6. Reinigung von Hexahistidin-tragenden Hantavirus-Nukleokapsidproteinen Die sedimentierte unlösliche Proteinfraktion (siehe 4.) wird in 5 ml Qiagen-Puffer A gelöst ( 6 M Guanidinhydrochlorid; 0,1 M NaH2PO4; 0,01 M Tris-HCl, pH 8,0). Zwei ml M+-NTA- Resin, vorher äquilibriert in Puffer A, wird dazugegeben und anschließend 1 Stunde geschüttelt. Die Reinigung der N-Proteine erfolgt mit Hilfe einer Säulenchromatographie, entsprechend der Vorschrift des Herstellers für die denaturierende Reinigung von unlöslichen Proteinen (Qiagen, Hilden, Deutschland). Geringe Mengen der rekombinanten Proteine werden in Puffer D, aber der Hauptteil der N-Proteine wird in Puffer E (8 M Harnstoff; 0, 1 M NaH2PO4; 0,01 M Tris-HCl, pH 4,5) eluiert. Fraktionen werden gesammelt und anschließend mittels SDS-Polyacylamid-Gelelektrophorese und Western blot analysiert. Die 3 rekombinanten Proteine wurden mit einer Ausbeute von im Durchschnitt etwa 1,25 mg pro g Feuchtgewicht (entspricht ca. 15 mg pro 1 Kulturvolumen) erhalten.6. Purification of hexahistidine-bearing Hantavirus nucleocapsid proteins The sedimented insoluble protein fraction (see 4.) is dissolved in 5 ml Qiagen buffer A (6 M guanidine hydrochloride; 0.1 M NaH 2 PO 4 ; 0.01 M Tris-HCl, pH 8.0). Two ml of M + -NTA resin, previously equilibrated in buffer A, is added and then shaken for 1 hour. The N proteins are purified using column chromatography, in accordance with the manufacturer's instructions for the denaturing purification of insoluble proteins (Qiagen, Hilden, Germany). Small amounts of the recombinant proteins are in buffer D, but the majority of the N proteins are eluted in buffer E (8 M urea; 0.1 M NaH 2 PO 4 ; 0.01 M Tris-HCl, pH 4.5). Fractions are collected and then analyzed by SDS-polyacylamide gel electrophoresis and Western blot. The 3 recombinant proteins were obtained with an average yield of approximately 1.25 mg per g wet weight (corresponds to approximately 15 mg per 1 culture volume).
7. Schutz von Rötelmäusen gegen Hantavirus-Challenge durch Immunisierung mit authentischem, Hefe-exprimiertem Puumalavirus-Nukleokapsidprotein7. Protection of Rubella against Hantavirus Challenge by Immunization with Authentic, Yeast-Expressed Puumala Virus Nucleocapsid Protein
Entsprechend einem Standard-Protokoll (Lundkvist et al., 1996, Virology 216, 397-406; Ulrich et al., 1998, Vaccine 16, 272-280) werden Rötelmäuse, Clethrionomys glareolus, mit Nickelchelatchromatografie-gereinigtem PUUV-His-N-Protein immunisiert. Entsprechend diesem Protokoll werden 3 Immunisierungen mit jeweils 50 μg im Abstand von jeweils 3 Wochen subkutan verabreicht. Die erste Immunisierung erfolgt unter Zusatz von komplettem Freund's Adjuvanz, die zweite mit inkomplettem Freund's Adjuvanz, die dritte ohne zusätzliches Adjuvanz. Das Virus-Challenge erfolgt durch Inokulation mit 104 ID o Einheiten des PUUV-Stammes Kazan (Lundkvist et al, 1997, J. Virol. 71, 9515-9523). Drei Wochen nach Challenge werden die Tiere bezüglich der Infektionsmarker analysiert. Das Fehlen von N-Antigen und S-Segment-spezifischer RNA in der Lunge (bestimmt mit Hilfe der PCR; Niklasson und Lundkvist, 1999, In: Manual of Hemorrhagic Fever with renal syndrome and hantavirus pulmonary syndrome; H.W. Lee, C. Calisher, C. Schmaljohn, Hrg.; WHO Collaborating Center for Virus Reference and Research, Asian Institute for Life Sciences, Seoul) sowie von G2-spezifischen Antikörpern im Serum (nach Challenge) zeigt eine vollständige sterile Protektion an, während der Nachweis einzelner Infektionsparameter auf eine partielle Protektion hinweist. Alle 4 immunisierten Tiere zeigen keinen der Infektionsmarker an, d.h. sie werden durch die Immunisierung vollständig vor dem Viruschallenge geschützt (Tab. 1). In nicht- vakzinierten Tieren werden nach Challenge alle 3 Infektionsmarker nachgewiesen; in Tieren ohne Challenge fehlen alle 3 Infektionsmarker.According to a standard protocol (Lundkvist et al., 1996, Virology 216, 397-406; Ulrich et al., 1998, Vaccine 16, 272-280), rubella mice, Clethrionomys glareolus, with PUUV-His-N- purified with nickel chelate chromatography. Protein immunized. According to this protocol, 3 immunizations, each with 50 μg, are administered subcutaneously every 3 weeks. The first immunization is carried out with the addition of complete Freund's adjuvant, the second with incomplete Freund's adjuvant, the third without additional adjuvant. 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.
In einem zweiten, unabhängigen Immunisierungs/Challenge-Experiment unter Verwendung des Nickelchelatcliromatografie-gereinigten PUUV-His-N-Proteins konnte für 8 weitere Rötelmäuse eine vollständige Protektion gezeigt werden.In a second, independent immunization / challenge experiment using the nickel chelate chromatography purified PUUV-His-N protein, complete protection was demonstrated for 8 other rubella mice.
Tabelle 1 : Nachweis der Induktion einer protektiven Immunantwort in RötelmäusenTable 1: Evidence of the induction of a protective immune response in rubella
Figure imgf000013_0001
Figure imgf000013_0001
vollständige Protektion; n.d. - nicht durchgeführt 8. Schutz von Rötelmäusen gegen Hantavirus-Challenge durch Immunisierung mit Hefe-exprimiertem Puumalavirus-Nukleokapsidprotein unter Verwendung eines für humane Anwendung zugelassenen Adjuvanz (Aluminiumhydroxid)full protection; nd - not performed 8. Protection of Rubella against Hantavirus Challenge by Immunization with Yeast-Expressed Puumala Virus Nucleocapsid Protein Using an Adjuvant Approved for Human Use (Aluminum Hydroxide)
Entsprechend dem unter 7. genannten Standardprotokoll werden Rötelmäuse mit Nickelchelatchromatografie-gereinigtem PUUV-His-N-Protein unter Verwendung von Aluminiumhydroxid immunisiert. Dabei werden 3 Immunisierungen mit jeweils 50 μg im Abstand von jeweils 3 Wochen subkutan verabreicht. Die ersten beiden Immunisierungen erfolgen unter Zusatz von Aluminiumhydroxid und die dritte Immunisierung unter Verwendung von PBS. Das Virus-Challenge erfolgt wie unter 7. beschrieben. Das FehlenAccording to the standard protocol mentioned under 7, rubella mice are immunized with PUUV-His-N protein purified by nickel chelate chromatography using aluminum hydroxide. 3 immunizations with 50 μg 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
1) von G2-spezifischen Antikörpern nach Challenge,1) of G2-specific antibodies after challenge,
2) einer Erhöhung des Levels N-Protein-spezifischer Antikörper nach Challenge sowie 3) von S-Segment-spezifischer RNA in der Lunge zeigt eine vollständige sterile Protektion an, während der Nachweis einzelner Protektionsparameter auf einen partiellen Schutz hinweist. Alle 8 immunisierten Tiere zeigen durch das Fehlen von G2-spezifischen Antikörpern und einer Erhöhung des Levels N-Protein- spezifischer Antikörper nach Challenge zumindest einen partiellen Schutz (Tabelle 2). 6 von 8 Tieren sind vollständig gegen den Challenge geschützt, da sie alle 3 genannten Marker der Protektion zeigen. In zweien der 8 Tiere ist S-Segment-spezifische RNA nachweisbar (partieller Schutz). 2) an increase in the level of N-protein-specific antibodies after challenge, and 3) an S-segment-specific RNA in the lungs indicates complete sterile protection, while the detection of individual protection parameters indicates partial protection. All 8 immunized animals show at least partial protection due to the lack of G2-specific antibodies and an increase in the level of N-protein-specific antibodies after challenge (Table 2). 6 out of 8 animals are completely protected against the challenge because they show all 3 mentioned markers of protection. S-segment-specific RNA can be detected in two of the 8 animals (partial protection).
Tabelle 2: Nachweis der Induktion einer protektiven Immunität durch Immunisierung mit PUUV-His-N-Protein unter Verwendung von Aluminiumhydroxid als AdjuvanzTable 2: Evidence of the induction of protective immunity by immunization with PUUV-His-N protein using aluminum hydroxide as adjuvant
Figure imgf000015_0001
Figure imgf000015_0001
* Optische Dichte nd - nicht durchgeführt; positive Kontrolle - nicht-immunisierte Tiere mit Virus-Challenge; negative Kontrolle - nicht-immunisierte Tiere ohne Virus-Challenge. Abkürzungsverzeichnis* Optical density nd - not carried out; positive control - non-immunized animals with virus challenge; negative control - non-immunized animals without virus challenge. List of abbreviations
ATCC American Type Culture Collection DOBV Hantavirus-Serotyp DobravaATCC American Type Culture Collection DOBV Hantavirus serotype Dobrava
DNA Desoxyribonukleinsäure (desoxyribonucleic acid)DNA deoxyribonucleic acid (deoxyribonucleic acid)
E. coli Escherichia coliE. coli Escherichia coli
EDTA EthylendiamintetraessigsäureEDTA ethylenediaminetetraacetic acid
ELISA Enzyme Linked Immunosorbent Assay EP Europäische PatentanmeldungELISA Enzyme Linked Immunosorbent Assay EP European patent application
FDH1 Gen von Candida maltosa, das eine Formaldehyddehydrogenase kodiertFDH1 gene from Candida maltosa that encodes a formaldehyde dehydrogenase
(vermittelt Resistenz gegen Formaldehyd)(imparts resistance to formaldehyde)
Gl Hantavirus-Glykoprotein GlGl hantavirus glycoprotein Gl
G2 Hantavirus-Glykoprotein G2 GAL10-PYK1 Hefe-spezifischer Hybrid-PromoterG2 Hantavirus glycoprotein G2 GAL10-PYK1 yeast-specific hybrid promoter
HBsAg HBV-OberflächenantigenHBsAg HBV surface antigen
HBV Hepatitis-B- VirusHBV hepatitis B virus
HFRS Hämorrhagisches Fieber mit Renalem SyndromHFRS Hemorrhagic Fever with Renal Syndrome
HPS Hantavirales Pulmonales Syndrom HTNV Hantavirus-Serotyp HantaanHPS Hantavirales Pulmonary Syndrome HTNV Hantavirus serotype Hantaan
ID50 Infektionsdosis (Prozentsatz) L3 Labor der Sicherheitsstufe 3ID 50 infection dose (percentage) L3 Laboratory of security level 3
Ni+-NTA Nickel-Nitrilotriacetic acid PBS Phosphatpuffer (phosphate buffered saline) PCR Polymerase-Kettenreaktion (polymerase chain reaction) PCT Patent Cooperation Treaty pFX7 Hefeexpressionsplasmid PGK1 Gen von Saccharomyces cerevisiae, das eine Phosphoglyceratkinase kodiert PMSF Phenylmethylsulfonylfluorid PS PatentschriftNi + -NTA nickel-nitrilotriacetic acid PBS phosphate buffered saline PCR polymerase chain reaction PCT Patent Cooperation Treaty pFX7 yeast expression plasmid PGK1 gene from Saccharomyces cerevisiae, which encodes a phosphoglycerate kinase PMSF phenylmethylsulfonyl
PUUV Hantavirus-Serotyp PuumalaPUUV Hantavirus serotype Puumala
RNA Ribonucleinsäure (ribonucleic acid)RNA ribonucleic acid
SDS Natriumdodecylsulfat (Sodium Dodecylsulfat) SEOV Serotyp SeoulSDS sodium dodecyl sulfate (sodium dodecyl sulfate) SEOV serotype Seoul
Tris Tris-(hydroxymethyl)-aminomethanTris tris (hydroxymethyl) aminomethane
Tween 20 Tween . ® : RÖMPP CHEMIE LEXIKON, Georg Thieme Verlag StuttgartTween 20 tween. ® : RÖMPP CHEMIE LEXIKON, Georg Thieme Verlag Stuttgart
New York 1995, Tween 20: Polyethoxysorbitanlaurat VACV Vaccinia VirusNew York 1995, Tween 20: Polyethoxysorbitan laurate VACV Vaccinia Virus
WO Internationale Patentanmeldung über PCT WHERE International patent application via PCT

Claims

Patentansprüche claims
1. Hantavirus-Impfstoffe, die als wirksame Komponenten rekombinante Hantavirusproteine enthalten, hergestellt in Hefezellen.1. Hantavirus vaccines, which contain recombinant hantavirus proteins as effective components, produced in yeast cells.
2. Impfstoffe nach Anspruch 1, dadurch gekennzeichnet, dass sie als wirksame Komponenten2. Vaccines according to claim 1, characterized in that they are effective components
2.1. Nukleokapsidproteine und/oder2.1. Nucleocapsid proteins and / or
2.2. Glykoproteine von Hantaviren enthalten.2.2. Contain hantavirus glycoproteins.
3. Impfstoffe nach Anspruch 1 und 2, dadurch gekennzeichnet, dass sie Nukleokapsidproteine der Hantavirus-Serotypen Puumala, Dobrava und/oder Hantaan enthalten.3. Vaccines according to claim 1 and 2, characterized in that they contain nucleocapsid proteins of the Hantavirus serotypes Puumala, Dobrava and / or Hantaan.
4. Impfstoffe nach Anspruch 1 bis 3, dadurch gekennzeichnet, dass sie Nukleokapsidproteine der Hantavirus-Stämme Puumala-Vranica/Hällnäs, Dobrava-Slovakia und/oder Hantaan-4. Vaccines according to claims 1 to 3, characterized in that they contain nucleocapsid proteins from the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan-
Fojnica enthalten.Fojnica included.
5. Impfstoffe nach Anspruch 1 bis 4, dadurch gekennzeichnet, dass als Hefezellen Saccharomyces cerevisiae Verwendung finden.5. Vaccines according to claim 1 to 4, characterized in that Saccharomyces cerevisiae are used as yeast cells.
6. Impfstoffe nach Anspruch 1 bis 5, dadurch gekennzeichnet, dass als Hefezellen Saccharomyces cerevisiae, Stamm fh4c, verwendet wird.6. Vaccines according to claims 1 to 5, characterized in that Saccharomyces cerevisiae, strain fh4c, is used as the yeast cell.
7. Verfahren zur Herstellung von Hantavirus-Impfstoffen der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass man als wirksame Komponenten rekombinante Hantavirusproteine einsetzt, die in Hefezellen gewonnen werden.7. A process for the preparation of hantavirus vaccines of claims 1 to 6, characterized in that recombinant hantavirus proteins are used as effective components, which are obtained in yeast cells.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, dass als wirksame Komponenten Nukleokapsidproteine und/oder Glykoproteine 8.1. von Hantaviren8. The method according to claim 7, characterized in that the active components are nucleocapsid proteins and / or glycoproteins 8.1. of hantaviruses
8.2. der Hantavirus-Serotypen Puumala, Dobrava und/oder Hantaan8.2. the Hantavirus serotypes Puumala, Dobrava and / or Hantaan
8.3. der Hantavirus-Stämme Puumala-Vranica/Hällnäs, Dobrava-Slovakia und/oder Hantaan- Fojnica eingesetzt werden.8.3. the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan- Fojnica be used.
9. Verfahren nach den Ansprüchen 7 und 8, dadurch gekennzeichnet, dass als Hefezellen Saccharomyces cerevisiae, insbesondere der Stamm fh4c, eingesetzt wird.9. The method according to claims 7 and 8, characterized in that Saccharomyces cerevisiae, in particular the strain fh4c, is used as yeast cells.
10. Verfahren nach den Ansprüchen 7 bis 9, dadurch gekennzeichnet, dass ein Hefeexpressionsplasmid eingesetzt wird, das einen Hefe-spezifischen GALIO-PYKI-Hybrid- Promoter und das FDHl-Gen von Candida maltosa als dominanten Selektionsmarker trägt.10. The method according to claims 7 to 9, characterized in 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.
11. Verfahren nach den Ansprüchen 7 bis 10, dadurch gekemizeichnet, dass die Hefezellen in YEPD-Medium (1% Hefe-Extrakt, 2%Pepton, 2% Glukose) mit 5 mM Formaldehyd angezüchtet werden und durch Zugabe von Galaktose (Endkonzentration 3%) die Synthese der Hantavirusproteine induziert wird.11. The method according to claims 7 to 10, characterized in that the yeast cells are grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) with 5 mM formaldehyde and by adding galactose (final concentration 3% ) the synthesis of the hantavirus proteins is induced.
12. Verfahren nach den Ansprüchen 7 bis 11, dadurch gekennzeichnet, dass die Hefezellen durch Glasperlen aufgeschlossen werden.12. The method according to claims 7 to 11, characterized in that the yeast cells are broken down by glass beads.
13. Verfahren nach den Ansprüchen 7 bis 12, dadurch gekennzeichnet, dass die rekombinanten Hantavirusproteine durch zwei aufeinanderfolgende Cäsiumchlorid- Dichtegradienten-Zentrifugationen oder durch Anreicherung der unlöslichen Proteine und anschließende Nickelchelat-Affinitätschromatografie gereinigt werden.13. The method according to claims 7 to 12, characterized in that 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.
14. Verwendung der rekombinanten Hantavirusproteine nach Anspruch 1 als wirksame Komponenten in Hantavirus-Impfstoffen.14. Use of the recombinant hantavirus proteins according to claim 1 as effective components in hantavirus vaccines.
15. Verwendung nach Anspruch 14, dadurch gekennzeichnet, dass Nukleokapsidproteine und/oder Glykoproteine15. Use according to claim 14, characterized in that nucleocapsid proteins and / or glycoproteins
15.1. von Hantaviren15.1. of hantaviruses
15.2. der Hantavirus-Serotypen Puumala, Dobrava und/oder Hantaan 15.3. der Hantavirus-Stämme Puumala-Vranica/Hällnäs, Dobrava-Slovakia und/oder Hantaan- Fojnica als wirksame Komponenten in Hantavirus-Impfstoffen eingesetzt werden. 15.2. the Hantavirus serotypes Puumala, Dobrava and / or Hantaan 15.3. of the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan-Fojnica are used as effective components in Hantavirus vaccines.
16. Verwendung einer nach den Ansprüchen 1 bis 13 hergestellten Vakzine zur Induktion einer protektiven Immunität.16. Use of a vaccine produced according to claims 1 to 13 for the induction of protective immunity.
17. Verwendung mach Anspruch 16 zur Induktion einer protektiven Immunität durch Immunisierung mit PUUV-His-N-Protein unter Verwendung von Aluminiumhydroxid als17. Use make claim 16 for the induction of a protective immunity by immunization with PUUV-His-N protein using aluminum hydroxide as
Adjuvanz.Adjuvant.
18. Verwendung von Hefezellen nach den Ansprüchen 1, 5 und 6, insbesondere Saccharomyces cerevisiae, vorzugsweise des Stammes fh4c, zur Herstellung rekombinanter Hantavirusproteine.18. Use of yeast cells according to claims 1, 5 and 6, in particular Saccharomyces cerevisiae, preferably of the strain fh4c, for the production of recombinant hantavirus proteins.
19. Verwendung der rekombinanten Hantavirusproteine nach den Ansprüchen 1 bis 4 in der Diagnostikaentwicklung. 19. Use of the recombinant hantavirus proteins according to claims 1 to 4 in diagnostic development.
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