DE10055886A1 - Vaccines containing recombinant hantavirus proteins, processes for their preparation and their use - Google Patents

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

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 said to be the highly efficient production of non-infectious, endotoxin-free recombinant Hantavirus proteins make it suitable for human use as a vaccine are.

Hantaviruses are becoming the cause of their increasing importance as pathogens "emerging viruses" counted. They are the pathogen that occurs in Europe and Asia Hemorrhagic Fever with Renal Syndrome, HFRS, (Schmaljohn et al., 1985, Science 227, 1041-1044) and the new one that appeared in America in the early 1990s, with high Lethality-related Hantaviral Pulmonary Syndrome, HPS (Nichol et al., 1993, Science 262, 914-917). That as HFRS (list of abbreviations behind The clinical picture summarized in the exemplary embodiments) is different Serotypes caused: The most serious course of the disease is caused by infections with the Serotypes Hantaan (HTNV) and Dobrava (DOBV), while infections with the serotypes Seoul (SEOV) and Puumala (PUUV) show milder clinical courses. Overall kick around 200,000 HFRS cases per year worldwide (Krüger and Zöller, 1996, in: Virusdiagnostik, Porstmann, T., ed., Blackwell Wissenschaftsverlag, Berlin Vienna Oxford, pp. 117-128.). In Europe was able to show the co-existence of the human pathogenic serotypes DOBV and PUUV (Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411; Sibold et al., 2001, J. Med. Virol., In press).

Hantaviruses form a separate genus within the Bunyaviridae family (Schmaljohn et al., 1995, Arch. Virol. Suppl. 10, 300-315). The negative polarity RNA genome settles composed of 3 segments, the viral RNA polymerase, the glycoproteins G1 and G2 and encode the nucleocapsid protein (N).

Because of the endemic spread of Hantaviruses in Asia have been in China, South and North Korea and various inactivated hantavirus vaccines manufactured in Japan and tried (overview in Schmaljohn, 1994, Rev. Med. Virol. 4, 185-196). So far, only one HTNV dead vaccine (trade name: Hantavax) in Korea for human use  been approved (Cho and Howard, 1999, Vaccine 17, 2569-2575). Although with the so far developed whole virus dead vaccines e.g. Hopeful vaccination successes could be achieved, A number of open problems remain: Because of the high risk potential the hantaviruses must reproduce under L3 laboratory conditions. The Virus yield when cultivated in cell cultures is very low. The ones tested so far Low virus neutralizing activity may be observed by vaccines the only small proportion of G1 and G2 in the total protein and the destruction of the G1 and G2 conformation caused by virus inactivation.

One solution to the problems mentioned is the production of recombinant Virus subunit vaccines. In animal experiments, immunization with Vaccinia Virus (VACV)-derived vaccines that cover the complete M segments of HTNV or SEOV, a protective immune response against the corresponding homologous virus (Schmaljohn et al., 1990, J. Virol. 64, 3162-3170; Xu et al., 1992, Am. J. Trop. Med. Hyg. 47, 397-404) can be induced. While immunized with HTNV- and SEOV-VACV- Recombinant was also protected against SEOV and HTNV, was against a PUUV Challenge not protected (Chu et al., 1995, J. Virol. 69, 6417-6423).

A recombinant HTNV-VACV vaccine showed that in a clinical phase I study Immunogenicity and safety of the vaccine. A phase II study showed immunization in ¾ of VACV-naive subjects the induction of virus-neutralizing Antibodies, but only in about ¼ of the VACV-immune test subjects (McClain et al., 2000, J. Virol. 60, 77-85).

The previously produced naked DNA vaccines based on G1 / G2-encoding Constructs showed the induction of virus-neutralizing antibodies and in animal models Protection to (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, with inoculation no or only weak protection is induced by N-coding constructs. However, there are still a number of questions for the clinical application of DNA vaccines regarding safety (risk of tolerance induction, integration of DNA, Autoimmunity) (Davis and Cluskie, 1999, Microbes and Infection 1, 7-21). Alternatively, Sindbis virus-derived constructs were tested, but one clearly showed less protection than DNA vaccine constructs (Kamrud et al., 1999).

Compared to the previously mentioned systems, recombinant proteins offer in terms of Security clear advantages (no infectivity, no integration). In a series of  heterologous expression systems, such as E. coli and insect cells, could be recombinant Hantavirus proteins are made that have a protective immune response in animal models induced (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). During E. coli expression systems, high expression of Hantavirus proteins allow possible contamination with bacterial Endotoxins pose a problem for human use. In contrast, offer However, insect cell expression systems have the advantage of being free of endotoxins inferior to the E. coli systems in their synthesis performance for foreign proteins.

The history of the discovery and control of hantaviruses can also be illustrated document the patent literature. C. S. Schmaljohn and J. Dalrymple initially had 1987 and then in 1991 filed with D. J. McClain in the United States patent applications that refer to hantavirus proteins or vaccines, in particular to the glycoproteins G1 and G2: US Pat. No. 5,298,423 and 5,614,193. In 1993, S. T. Nichol, C. F. Spiropoulou, T. G. Ksiazek and P. E. Rollin for the detection of the Hantavirus "Four Corners Protect virus "(US 5945277, WO 9500648), 1995 from this group P.E. Rollin, L. Elliot, T. G. Ksiazek and S. T. Nichol the detection of the "Black Creek Canal Hantavirus" (US 5853980). In 1996, H. S. Kim, W. D. Yoo, S. O. Kim, K. S. Noh and S. P. Hong reported in Korea "Hantaan virus nucleocapsid protein expressed from E. coli" (WO 9727302) for a patent, the same year in Sweden (SE) Å. Lundkv is a vaccine against Hantavirus or Puumala virus based on the nucleocapsid protein (N) (WO 9728819). In the USA followed in 1997 by B. L. Hjelle and N. Torrez-Martinez with a nucleocapsid protein vaccine from "Rio Mamore Hantavirus" (WO 9901153), 1998 in Korea H. W. Lee, S. J. Ryu, C. N. Ahn, H. Kim and T. Tomiyama with an application for the title "Diagnostic reagent for Puumala virus infection "(EP 952450). The United States applications go from 1999 to S. T. Nichol, S. Morzunov, T.G. Ksiazek and P.E. Rollin on the discovery and isolation of the new Hantavirus "Bayou" (US 5916754) and by C. S. Schmaljohn and J. W. Hooper on a DNA vaccine against hantavirus infections (WO 2000044406). The shown However, solutions leave the problems of recombinant proteins already mentioned above from E. coli, from DNA vaccines and VACV-derived live vaccines.

The invention has for its object to develop new hantavirus vaccines for human applications are harmless. The task was solved by using a yeast expression system that allows high expression of hantavirus proteins,  recombinant hantavirus proteins isolated in large quantities from yeast cells and by simple Centrifugation steps and / or purified by affinity chromatography. The Proteins obtained according to the invention have proven to be endotoxin-free and for human Applications proven safe.

The invention has the PCR amplification and cloning of hantavirus protein coding sequences to the content. These DNA sequences are in Yeast expression vectors with a yeast-specific expression unit (promoter and Transcription stop signal) installed. The expression of the hantavirus proteins takes place in Yeast cells, preferably in Saccharomyces cerevisiae strain fh4c (ATCC # 42368; Colona and Lampen, 1974, Biochemistry 13, 2741-2748). After opening the yeast cells with the help of Glass beads become the authentic Hantavirus proteins by two successive ones Cesium chloride density centrifugations, hexahistidine-carrying hantavirus proteins by Enrichment of the insoluble protein and subsequent nickel chelate chromatography cleaned. 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. The protection of the proteins is determined by Immunization of rubella, the natural host of the PUUV, and subsequent PUUV load (challenge) provided. Protection is based on the lack of anti-G2 Antibodies in the serum, lack of N antigen in the lungs and lack of RNA detection performed using S-segment-specific PCR.

The invention makes a new expression system for hantavirus proteins available, this has significant advantages over the previous systems in E. coli or insect cells having. So yeast expression systems allow a 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 for human The following are approved for vaccine production: The recombinant HBV vaccine is based on yeast expressed HBsAg.

It is particularly surprising that the yeast cells contain the hantavirus proteins in large quantities synthesize. In addition, the recombinant proteins after lysis of the yeast cells in large quantities can be isolated and cleaned using simple methods. By the nature of the Obtaining from yeast cells, the proteins according to the invention are endotoxin-free and for humane applications suitable as vaccines and in the development of diagnostics.  

The essence of the invention is the provision of hantavirus vaccines, which as effective components contain recombinant hantavirus proteins, the proteins in turn are produced in yeast cells. They contain as effective components Hantavirus nucleocapsid proteins and / or glycoproteins, such as the Hantavirus serotypes Puumala, Dobrava and / or Hantaan, in particular the Hantavirus strains Puumala- Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan-Fojnica. Find as yeast cells Saccharomyces cerevisiae use, especially of the strain fh4c.

A special feature of the method according to the invention is that a Yeast expression plasmid is used, which contains a yeast-specific GAL10-PYK1 hybrid Promoter and the FDH1 gene from Candida maltosa as the dominant selection marker. The yeast cells are in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) with 5 mM Formaldehyde grown, then by adding galactose (final concentration 3%) induced the synthesis of the hantavirus proteins. The yeast cells are disrupted through glass beads.

The recombinant hantavirus proteins are replaced by two consecutive ones Cesium chloride density gradient centrifugations or by enriching the insoluble ones Proteins and subsequent nickel chelate affinity chromatography purified.

The uses of the recombinant hantavirus proteins according to the invention are in their use as effective components in hantavirus vaccines as well as in the Diagnostics development based on the ELISA and / or immunoblot principle.

The features of the invention go beyond the claims and also from the description , the individual features each individually or in groups in the form of Combinations represent advantageous protective designs for those with this font Protection is requested. The combination consists of known (hantavirus proteins, Centrifugation, affinity chromatography) and new elements (a novel Expression system for hantavirus proteins) which influence each other and in their new overall effect give a benefit in use and the desired success, which is in it lies that now recombinant Hantavirus proteins in large quantities from yeast cells isolated, cleaned by simple work steps (centrifugation, chromatography) and the Can be made available to users.

The invention is to be explained in more detail below using an exemplary embodiment, without to be limited to this example.  

embodiment 1. Obtaining the hantavirus coding sequences

The complete open reading frames for PUUV- (strain Vranica / Hällnäs; Reip et al., 1995, Arch. Virol. 140, 2011-2026), DOBV- (Slovakian tribe 862; Sibold et al., 2001, J. Med. Virol., In press) and HTNV- (strain Fojnica; Sibold et al., 1999, Am. J. Trop. Med. Hyg. 61, 409-411) N proteins are PCR-amplified using standard methods of the following primers that carry an XbaI restriction site:

PUUV (Vranica / Hällnäs; Fig. 1)

Forward primer

Reverse primer

DOBV (Slovakia 862; Fig. 2)

Forward primer

Reverse primer

HTNV (Fojnica; Fig. 3)

Forward primer

Reverse primer

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-Vranica N, pCRII-Slovakia N and pCR-Blunt II-TOPO-Fojnica N are verified by DNA sequencing ( FIGS. 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-Vranica N, pCRII-Slovakia N and pCR-Blunt II-TOPO-Fojnica N with the following PCR amplified primers ( Fig. 1-3):

His-PUUV (Vranica / Hällnäs; Fig. 1)

Forward primer

Reverse primer

His-DOBV (Slovakia 862; Fig. 2)

Forward primer

Reverse primer

His-HTNV (Fojnica; Fig. 3)

Forward primer

Reverse primer

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 ( Fig. 1 to 3).

Fig. 1 to 3
PCR amplification and cloning of the hantavirus N protein coding sequences

Legend to FIGS. 1 to 3

Fig. 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 Protein

Fig. 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 Protein

2. Preparation of yeast expression vectors

The yeast expression plasmid, preferably pFX7 (Scherneck, Sasnauskas and Ulrich, published application DE 197 50 220 A1, PCT / DE 98/03329; Sasnauskas et al., 1999, Biol. Chem. 380, 381-386; see Fig. 4)
a yeast-specific GAL10-PYK1 hybrid promoter,

• A transcription termination sequence (from PGK1),
• A 2 µm DNA fragment,
• - the FDH1 gene from Candida maltosa as the dominant selection marker
• - And a unique restriction site for the restriction endonuclease XbaI for 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 XbaI or SpeI cleavages and inserted into the yeast expression vector pFX7 linearized with XbaI (in FIG. 5, FIG. 6 is as Example of the construction of the pFX7-derived plasmids with PUUV-N coding inserts shown). After transformation into E. coli K12, recombinant plasmids are selected and characterized by restriction mapping.

Fig. 4 to 6
Construction of the pFX7-derived expression plasmids

Fig. 4: pFX7

Fig. 5: pFX7-PUUV-N

Figure 6: pFX7-His ₆ -PUUV-N

3. Expression of Hantavirus nucleocapsid proteins in yeast

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% glucose; with 5 mM formaldehyde) are inoculated with the yeast cells and cultivated for 24 hours at 30 ° C. with shaking. 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 removed and denatured with lysis buffer (0, 125 M Tris-HCl, pH 6.8; 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.2 mg / 10 ml bromophenol blue). 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).

Fig. 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- Negative control (pFX7).

4. Disruption of the yeast cells and enrichment of the hantavirus nucleocapsid proteins

After sedimentation, the yeast cells grown as described under 3. are resuspended in 10 ml 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 at 2000 xg for 5 minutes. Insoluble proteins are sedimented by centrifugation at 10,000 xg for 40 minutes (4 ° C). Since the recombinant hantavirus N proteins are found almost exclusively in the sediment, the supernatant is discarded. In order to further enrich the N-proteins in the pellet fraction, contaminating cellular proteins are extracted twice by resuspending the pellet in 10 ml of extraction buffer (20 mM PBS, pH 7.6; 2 mM EDTA; 1 mM PMSF; 1% Tween 20), Shake for 1 hour at 4 ° C and centrifugation at 10,000 xg 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 -etailed N-derivatives by nickel chelate affinity chromatography (see 6.).

5. Purification of the authentic Hantavirus nucleocapsid proteins

The sediment as described under 4. is resuspended in 4 ml extraction buffer. Two ml of the suspension are placed on 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; 2mM EDTA, 1% Tween 20) layered and 24 hours at 36000 rpm at 4 ° C (Beckman ultracentrifuge) centrifuged. Gradient fractions of 0.5 ml are separated by SDS Characterized polyacrylamide gel electrophoresis and Western blot analysis. Hantavirus Protein-containing fractions are subjected to a second cesium chloride density gradient Subjected to centrifugation. Positive gradient fractions are dialyzed against PBS, aliquoted and stored at -20 ° C. The yield of the 3 recombinant hantavirus proteins, the Bradford method (Bradford reagent from BioRad, Munich, Germany; Bradford, 1976, analyte. Biochem. 72, 248-254) was determined on average approx. 1.5 mg per g wet weight (corresponds to approx. 18 mg per 1 culture volume).

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 Ni + -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. Protection of rubella against Hantavirus challenge by immunization with authentic, yeast-expressed Puumala virus nucleocapsid protein

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 with 50 µg each 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 ₅₀ 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, ed.; WHO Collaborating Center for Virus Reference and Research, Asian Institute for Life Sciences, Seoul) as well as serum G2-specific antibodies (after Challenge) indicates complete sterile protection, while the detection of individual infection parameters points to one partial protection indicates. 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.

Table 1

Evidence of the induction of a protective immune response in rubella

List of abbreviations

ATCC American Type Culture Collection
DOBV Hantavirus serotype Dobrava
DNA deoxyribonucleic acid (deoxyribonucleic acid)
E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid
ELISA Enzyme Linked Immunosorbent Assay
EP European patent application
FDH1 gene from Candida maltosa that encodes a formaldehyde dehydrogenase
(imparts resistance to formaldehyde)
G1 Hantavirus glycoprotein G1
G2 Hantavirus glycoprotein G2
GAL10-PYK1 yeast-specific hybrid promoter
HBsAg HBV surface antigen
HBV hepatitis B virus
HFRS Hemorrhagic Fever with Renal Syndrome
HPS Hantavirales Pulmonary Syndrome
HTNV Hantavirus serotype Hantaan
ID ₅₀

Infection dose (percentage)
L3 laboratory of security level 3
Ni + -NTA nickel-nitrilotriacetic acid
PBS phosphate buffered cream
PCR polymerase chain reaction
PCT Patent Cooperation Treaty
pFX7 yeast expression plasmid
PGK1 gene from Saccharomyces cerevisiae encoding a phosphoglycerate kinase
PMSF phenylmethylsulfonyl fluoride
PS patent specification
PUUV Hantavirus serotype Puumala
RNA ribonucleic acid
SDS sodium dodecyl sulfate (sodium dodecyl sulfate)
SEOV serotype Seoul
Tris tris (hydroxymethyl) aminomethane
Tween 20 tween. .®: RÖMPP CHEMIE LEXIKON, Georg Thieme Verlag Stuttgart
New York 1995, Tween 20: Polyethoxysorbitan laurate
VACV Vaccinia Virus
WHERE International patent application via PCT

Claims (17)

1. Hantavirus vaccines, which are effective components of recombinant hantavirus proteins contained, produced in yeast cells. 2. Vaccines according to claim 1, characterized in that they are effective components

• 1. 2.1. Nucleocapsid proteins and / or
• 2. 2.2. glycoproteins

of hantaviruses included. 3. Vaccines according to claim 1 and 2, characterized in that they are nucleocapsid proteins of the Hantavirus serotypes Puumala, Dobrava and / or Hantaan included. 4. Vaccines according to claims 1 to 3, characterized in that they contain nucleocapsid proteins of the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan- Fojnica included. 5. Vaccines according to claim 1 to 4, characterized in that as yeast cells Saccharomyces cerevisiae find use. 6. Vaccines according to claim 1 to 5, characterized in that as yeast cells Saccharomyces cerevisiae, strain fh4c, is used. 7. A process for the preparation of hantavirus vaccines of claims 1 to 6, characterized characterized in that as effective components recombinant hantavirus proteins uses, which are obtained in yeast cells. 8. The method according to claim 7, characterized in that the active components are nucleocapsid proteins and / or glycoproteins

• 1.1.1. of hantaviruses
• 2. 8.2. the Hantavirus serotypes Puumala, Dobrava and / or Hantaan
• 3. 8.3. the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan- Fojnica

be used. 9. The method according to claims 7 and 8, characterized in that as yeast cells Saccharomyces cerevisiae, in particular the strain fh4c, is used. 10. The method according to claims 7 to 9, characterized in that a Yeast expression plasmid is used, which contains a yeast-specific GAL10-PYK1 hybrid Promoter and the FDH1 gene from Candida maltosa as the dominant selection marker. 11. The method according to claims 7 to 10, characterized in that the yeast cells in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) with 5 mM formaldehyde be grown and the synthesis by adding galactose (final concentration 3%) the hantavirus protein is induced. 12. The method according to claims 7 to 11, characterized in that the yeast cells be broken down by glass beads. 13. The method according to claims 7 to 12, characterized in that the recombinant hantavirus proteins by two consecutive cesium chloride Density gradient centrifugation or by enriching the insoluble proteins and subsequent nickel chelate affinity chromatography. 14. Use of the recombinant hantavirus proteins according to claim 1 as effective Components in hantavirus vaccines. 15. Use according to claim 14, characterized in that nucleocapsid proteins and / or glycoproteins

• 1. 15.1. of hantaviruses
• 2. 15.2. the Hantavirus serotypes Puumala, Dobrava and / or Hantaan
• 3. 15.3. the Hantavirus strains Puumala-Vranica / Hällnäs, Dobrava-Slovakia and / or Hantaan- Fojnica

can be used as effective components in hantavirus vaccines. 16. 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 Hantavirusproteine. 17. Use of the recombinant hantavirus proteins according to claims 1 to 4 in the Diagnostics development.