DK175515B1 - DNA and RNA molecules derived from the western subtype of FSME virus, corresponding vectors, vaccinia expression vector, carrier bacterium, cell culture, peptide or polypeptide, preparation comprising peptides or polypeptides, 1 ... - Google Patents

Description

in DK 175515 B1

The present invention relates to DNA and RNA molecules of the western subtype of FSME virus and polypeptides encoded by these molecules, vectors, a carrier, a cell culture, a peptide or polypeptide, a preparation of one or more of these peptides or polypeptides, a live vaccine, its use, a diagnostic agent, and a DNA or RNA probe.

The western subtype of the spring summer meningoencephalitis (FSME) virus is a member of the Flaviviridae family, which are spherical lipid-enclosed RNA viruses (Westaway et al., Intervirology 24, 183-192, 1985). The prototype for these viruses is yellow 10 fever virus. By definition, all flaviviruses are serologically related, as evidenced by hemagglutination inhibition tests. However, by cross-neutralization, the family can be subdivided into several serocomplexes (DeMadrid and Porterfield, J.Gen.Virol. 23, 91-96, 1974), which include viruses that are more closely related to each other than to members of other serocomplexes or to ungrouped ones. 15 flaviviruses. FSME virus is a member of the so-called "Tick-borne" serocomplexes, which also include Louping-III, Langat, Omsk hemorrhagic fever, Kyasanur Forest Disease and Negishi virus.

Furthermore, FSME virus strains can be subdivided into a western (European) sub-type, transmitted mainly by Ixodes ricinus and a far-eastern type with Ixodes persulcatus as the major transmitter (Clarke, 1964, Bull. WHO 31, 45-56).

This subtype breakdown has been confirmed both by competitive radioimmunoassays as well as peptide mapping using limited proteolysis of the corresponding structural glycoproteins (Heinz and Kunz, J.Gen.Virol. 57, 263-274, 1981) and by antigen assays using monoclonal antibodies. (Heinz et al. Virology 126, 525-537, 1983).

In 30 mature virus particles, only three structural proteins, designated E, C and M, contain

In and have approximate molecular weights of 50,000-60,000, 15,000 and 7,000 respectively.

The genome of flaviviruses consists of single-stranded RNA with approx. 11,000 bases of I mRNA polarity with a molecular weight of 4x10 '/ 46 daltons. This RNA, in DK 175515 B1 together with the C protein, forms a spherical nucleocapsid surrounded by a lipid membrane associated with proteins E and M.

Experiments using purified preparations of the E protein obtained after solubilization of the virus with detergents have shown that E is the viral hemagglutinin (i.e., it causes agglutination of certain erythrocytes under suitable conditions) which after immunization, hemagglutin-H induces inhibitory, neutralizing and protective antibodies as well as immunity to live virulent virus infections (Heinz et al., Infect. Immun. 33, 250-1107, 1981).

In addition to these structural proteins, several virus-specific non-structural proteins can be found in flavivirus-infected cells.

Flavivira's genome organization has recently been determined by cDNA cloning and sequencing of yellow fever, West Nile and Murray Valley encephalitis virus (Rice et al., In Science 229, 726-733, 1985; Dalgarno et al., J.Mol. Biol. 187, 309-323, 1986; In Castle et al., Virology 147, 227-236, 1985; Castle et al., Virology 149, 10-26, 1986; Wengler et al., Virology 147, 264 -274, 1985). According to these analyzes,

20, flaviviras genome RNA maintains a single long open reading frame of ca. 11,000 Nucle- I

times that encode all structural and non-structural proteins.

The genes for the structural proteins are located in the 5 'portion of the RNA; they include approx.

In 1/4 of the genome in the order C-prM (M) -E. prM represents a precursor protein for M, which is present in immature forms of flavi viruses (Shapiro et al., Virology 56, 88-94, 1973). The proteolytic cleavage thereof, likely performed by a cellular protease found in the endoplasmic reticulum, leads to formation of M and evidently constitutes a late event during virus maturation. 1 35

It is believed that the translation of viral proteins is initiated and proceeds at the first or possibly the second AUG at the 5 'end of the RNA until it encounters a stop codon at the 3' end of the RNA. It is believed that the formation of single proteins occurs at a number of specific proteolytic cleavage events involving both cellular and virus-specific proteases.

DK 175515 B1 I

Pletnev et al. (FEBS 3660, 200, no. 2, 317-321, 1986) have cloned certain fragments.

ments of the Far Eastern subtype of the FSME virus and have demonstrated that genome organ I

the creation of this subtype of the FSME virus corresponds to the organization of the

above viruses, but they were unable to clone and sequence the entire sequence I

5 of the structural genes for this subtype of the FSME virus. Moreover, it has been found, I

that the published protein E sequence derived from the clone Sofjin 2 was not

correct because, due to a displacement of the reading frame, a wrong I was derived

sequence for the carboxy-terminal region. IN

10 So far, over 60 different flaviviruses are known, and about 2/3 of them are transmitted.

is carried by bite from an infected arthropod and thus constitutes "arthropod-borne" (ARBO) I

viruses. Several flaviviruses are known human pathogens, including yellow fever virus, Dengue-I

virus, Japanese encephalitis virus and FSME virus (Shope; in "The Togaviruses", p. 47- I

82, Academic Press, New York).

15 FSME viruses are endemic in many European countries, in Russia and China. The disease is well documented in some Central European countries such as Austria, Czechoslovakia or Hungary and every year hundreds of cases are admitted to hospitals, which is a significant public health problem.

2 °

The disease can be effectively prevented by vaccination with a highly purified, forma-I lin inactivated whole virus vaccine (Kunz et al., J. Med.Virol. 6, 103-109, 1980), which induces an immune response to the virus's structural proteins. The major disadvantage of this vaccine is that large quantities of infectious I25 and possibly dangerous virus suspensions must be handled during manufacture. Therefore, comprehensive and costly security measures are needed.

It is therefore an object of the present invention to provide means for preparing a vaccine to alleviate the aforementioned disadvantages.

In the present invention, this task is solved by providing an I DNA molecule comprising DNA derived from the western subtype of FSME virus, in which DNA encodes at least a portion of at least one protein selected from the group consisting of - At the end of proteins C, prM, M or E of the western subtype of FSME virus.

I 35 I DK 175515 B1 Η H The described DNA molecule corresponds to the single stranded RNA of the westernmost type of FSME virus and is suitable for providing genetic information for H expression of a polypeptide which may be the proteins C, prM, M, or E of the Western subtype of FSME virus, alone or in combination as described above, H5, or portions of one or more of the above proteins, which can be successfully used in H in diagnosis, therapy or prophylaxis.

Furthermore, the DNA molecule of the invention or parts thereof may be used for various purposes, as will be described in more detail below.

In the 10 H DNA molecule of the invention can be used as a whole molecule whose sequence H encodes the structural proteins of the western subtype of FSME virus, preferably as shown in FIG. 1. The DNA sequence shown in FIG. 1, the cloned genome of the western subtype of FSME virus exhibits the RNA molecule contained in the naturally occurring western subtype of FSME virus. The DNA sequence shown in FIG. 1, comprises the region of the structural proteins and the 5 'untranslated region. The DNA sequence examined has only one open reading frame; the amino terminal starting points of proteins C, prM, M and E as well as the I boundaries of clones A5 and P1-1 are shown by arrows.

As a preferred embodiment of the invention, DNA molecules are provided which encode a single of the Western protein subtype of FSME-I virus, i.e. for the entire C, prM, M or E protein. The respective portions of the DNA molecule of the invention can be used directly in suitable expression systems to prepare any of the desired structural proteins mentioned above.

It is a preferred object of the invention to provide a portion of the DNA molecule encoding at least the region of an antigenic determinant of one of the structural proteins of the western subtype of FSME virus for the production of diagnostic, therapeutically or prophylactically active agents. It is well known that regions of the protein's antigenic determinants are responsible for the induction of an antibody response. Therefore, not all the proteins for which they are known to have antigenic properties are necessarily used, but the antigenic determinant range of these proteins may be sufficient. It is known that in some cases a sequence 35 of only a few amino acids can act as antigen and cause an antibody response. Therefore, it may be sufficient to produce a vaccine to provide a DNA sequence which encodes only those amino acids that exhibit antigenic determinant functions. A DNA sequence encoding the antigenic determinant regions of the E-structural protein of the western subtype 5 of FSME virus is preferred.

There are two ways in which the DNA molecule of the invention can be prepared. One possibility by which the DNA molecule can be prepared is to first extract the viral RNA from the western subtype of FSME virus and then to purify the RNA molecule, followed by transcription of this RNA matrix into a DNA molecule under use of reverse transcriptase. Another option is to chemically synthesize the DNA molecule of the invention as soon as the DNA sequence is known.

As a preferred embodiment, the invention comprises DNA molecules that hybridize with the DNA molecules of the main claim under stringent conditions. DNA molecules of this preferred kind may also encode peptides capable of eliciting an antibody response, and in addition, these DNA molecules are useful as DNA probes.

The present invention expressly encompasses all of the DNA sequences that differ from the DNA molecules of the present invention, which are produced by degeneration of the genetic code and / or mutations and / or transpositions, but still encode a protein having the essential antigenic properties of at least one protein selected from the group consisting of proteins C, prM, M or E of the western subtype of FSME virus. For the above reasons, a DNA sequence different from that of FIG. 1, to a large extent diverge from DNA molecules made, isolated and purified in the form of cDNA by reverse transcriptase from an Western matrix of the FSME virus subtype. Nevertheless, the proteins encoded by this DNA molecule are still the same.

In a preferred embodiment of the invention, the DNA molecules of the invention can be combined with additional DNA sequences.

These additional sequences may enable replication and expression of the DNA molecule in a cell culture. To this end, the major DNA sequences originate from I DK 175515 B1 Η H enhancers, promoters, polyadenylation signals and splicing signals. These additional DNA sequences can be combined with the DNA molecules of the invention by standard methods known to those skilled in the art.

The advantages mentioned above for the DNA molecules of the invention also apply to RNA molecules. RNA molecules of the invention are peculiar in that they are derived from RNA from the Western subtype of FSME virus and encode

at least a portion of at least one protein selected from the proteins C, prM, M

H or E of the western subtype of FSME virus. In particular, the RNA molecule is one which encompasses the entire RNA sequence encoding the structural proteins, preferably as shown in FIG. 2. A particularly preferred variant is that it encodes one of the proteins selected from proteins C, prM, M, or E of the western subtype of FSME virus, or that it encodes at least one an area from an antigen

- determinant of one of the proteins selected from proteins C, prM, M or E

15 of the western subtype of FSME virus, preferably the E protein.

RNA molecules of the invention can be obtained by isolating and purifying RNA from the western subtype of FSME virus or by recombinant RNA / DNA techniques.

Furthermore, the invention encompasses not only RNA molecules obtained by purifying the western subtype of FSME virus, but also RNA molecules obtained by transcription of isolated and purified viral RNA into DNA by reverse transcriptase. and subsequent transcription of the DNA thus obtained into I RNA. A particularly preferred variant of an RNA molecule, which is characterized in that its complementary strand hybridizes under stringent conditions to an RNA molecule as set forth above.

In the present invention, not only the above-described DNA-I and RNA molecules are provided, but also vectors comprising the insertion of the subject DNA or RNA molecules, the vector being selected from plasmids, viruses and cosmic cells. . It is known to those skilled in the art that the vectors of the invention may comprise DNA sequences which control the replication and expression of the inserted RNA or DNA sequences such as, for example, promoters, enhancers, etc.

In a well-described and suitable plasmid for cloning of the DNA sequences, plasmid is pBR322. This plasmid contains a DNA sequence that encodes a protein selected from proteins C, prM, M or E of the western subtype of FSME virus or parts thereof.

A preferred plasmid prepared in the above manner is plasmid A5 shown in FIG. 5. Plasmid A5 is derived from plasmid pBR322 and contains the DNA sequence encoding the E protein. Another preferred plasmid is the plasmid P1.

The preferred plasmids referred to are deposited in the Deutsche Sammlung von Microorganism according to the Budapest Treaty and have the following deposit numbers: 10 a) plasmid A5 in E. coli HB101: DSM 4382 I b) plasmid Pl-1 in E. coli HB101: DSM 4383 By starting from the described plasmid A5, according to a preferred embodiment of the invention, insertion plasmids containing parts of the DNA sequence of the FSME virus cloned into plasmid A5 can be prepared, these insertion plasmids contains the foreign genetic information in such a way that, by homologous recombination, it transfers the DNA sequence of the E protein or parts I 20 thereof to a HindIII J fragment of vaccinia virus. Sub-fragments of plasmid A5I are cut with various restriction endonucleases and cloned into the insertion plasmids, for example, giving forms of the E protein which may or may not contain in hydrophobic amino acid sequences, i.e. signal and membrane anchoring sequences.

In Recombinant viruses with integrated protein E-DNA can be isolated from plaques. Integration of the FSME DNA sequences encoding desired portions of the E protein, I is confirmed by cutting with suitable restriction endonucleases and by hybridization I with 32P-labeled FSME cRNA and is visible by detection of a characteristic of HindIII J fragment from vaccinia. Three examples of plasmids suitable for carrying out recombination in vaccinia viruses are the plasmids pSC11-H1 containing the Haell fragment of the cloned I FSME DNA, the plasmid pSC11-F41 containing the FnuDII fragment of the cloned FSME DNA, as well as the plasmid pSC11-P6, which contains the PvuII fragment of the cloned FSME DNA. The plasmids described are shown in FIG. 7. The starting plasmid I 35 pSC11 is deposited in the Deutsche Sammlung von Microorganism according to I DK 175515 B1

I 8 I

Budapest Treaty in E. coli with the deposit number DSM 4381.

H The described insertion plasmids are preferably used for insertion of the desired sequences into vaccinia expression vectors. Thus, a preferred aspect of the invention is a vaccinia expression vector which, by homologous recombination, contains DNA sequences for the E protein, preferably from DNA sequences inserted into a plasmid according to any one of claims 22-25, and capable of expressing protein E or portions thereof containing the amino acid sequences corresponding to said DNA sequences.

I 10

As a carrier for the sequences, bacteria can also be used. A preferred bacterium is Salmonella typhi.

The vectors and carriers of the invention are preferably contained in cell cultures in which the expression of the polypeptides encoded by the RNA or DNA sequences of the invention takes place as far as possible under native conditions, with the cell culture being preferably a mammalian cell culture. . Using a mammalian cell culture, the most preferred conditions are provided for the expression of a polypeptide to be used as a vaccine.

I 20 - I Particularly suitable cell cultures for expression of the desired peptides or polypeptides are Vero cell cultures or chicken fetal fibroblast cell cultures containing I a vaccinia expression vector as described above, expressing the 1 I proteins corresponding to the in the vaccinia expression vectors contained DNA-I sequences. The recombinant vaccinia viruses can be assayed for the presence of the inserted FSME DNA by Southern Blot hybridization.

In accordance with the invention, there are provided peptides or polypeptides comprising amino acid sequences encoded by any of the above mentioned nucleotide sequences. The advantages of providing these peptides or polypeptides I are the same as the aforementioned advantages for the DNA and RNA molecules. 2 2

The amino acid sequences of the peptides or polypeptides of the invention correspond to the DNA or RNA sequences described above and are preferably the amino acid sequence shown in FIG. Third

DK 175515 B1 9

With the plasmids pSC11-H1, pSC11-F41 and pSC11-P6 described above, the expression of the proteins specified by them can preferably be performed after recombination in corresponding vaccinia expression vectors.

5

While it is preferred that the peptides or polypeptides of the invention be prepared by expression of one of the nucleotide sequences of the invention, especially in a cell culture as described above, it is also possible to chemically synthesize the peptides or polypeptides of the invention.

10

The peptides or polypeptides of the invention are preferably contained in a composition which can be used in the medical field.

The DNA and RNA sequences of the invention may also preferably be used to prepare a live vaccine; it is especially preferred that the DNA sequences be combined with DNA from vaccinia virus, which is well established as a live vaccine.

It is particularly preferred that the vaccine contains a vaccinia expression vector as defined above. On the other hand, a vaccine may be prepared containing a composition comprising one or more of the peptides or polypeptides encoded by the DNA or RNA sequences of the invention.

A further preferred use of vaccines containing antigenic peptides or polypeptides is to prepare specific immunoglobulins in a manner known to those skilled in the art.

25

The composition containing one or more of the peptides or polypeptides of the invention may also be used as a diagnostic agent.

The DNA or RNA sequences of the invention are also suitable as probes for identification purposes.

The invention is further explained by the following detailed description.

A preferred embodiment of the invention is shown in the drawing, in which FIG. Figure 1 shows the DNA sequence of the cloned genome of the western subtype of FSME virus, comprising the structural region and the 5 'untranslated region. The amino terminal starting points of proteins C, prM (M) and E as well as the boundaries of clones A5 and P1-1 are shown by arrows.

I.

FIG. Figure 2 shows the RNA sequence of the Western subtype of FSME virus encoding structural proteins. The amino terminal starting points of proteins C, prM (M) and E are shown by arrows.

FIG. 3 shows the amino acid sequence of the structural proteins of the western subtype of FSME virus, as derived from the one shown in FIG. 1. The NH2 terminals of the various proteins are shown by arrows.

FIG. 4 shows a photograph of an agarose gel electrophoresis of plasmids containing 15 insertions specific for the western subtype of FSME virus, after cutting with the restriction enzyme SmaI (lanes 2-6). The top band represents the plasmid DNA (4363 bp), the bottom band specific insertion DNA (in the range 2000 to 3500 bp). The DNA marker in lanes 1 and 7 corresponds to 23130, 9416, 6557, 4361, 2322, 2027, 564 and 125 base pairs (top and bottom).

In FIG. Figure 5 shows plasmid A5 derived from plasmid pBR322 and shown in restriction endonuclease intersection sites of selected sub-fragments, showing the corresponding regions encoding FSME protein E. The arrow shows the transcriptional direction of genomic RNA.

In Fig. 6, the insertion plasmid shows pSC11, which contains a single SmaI insertion site, the vaccinia promoter, the prokaryotic lacZ gene and vaccinia TK recombination sequences.

In FIG. Figure 7 shows three concrete examples of FSME insertion plasmids for in vivo Recombination in a vaccinia expression vector. The unique SmaI site at nucleotide I 6500 is shown in each plasmid.

In the first base of the FSME virus-specific sequences, in each of the plasmids shown, the nucleotide is 6503.

DK 175515 B1 11

FIG. 8 shows the characteristics of the subcloned FSME virus specific sequences. IN

The translation initiation codons, as well as hydrophobic and hydrophilic amino acid regions derived from a hydropathy plot, are shown. These areas were identified by 5 using an IBI-Pustell computer program.

Hydrophobic sequences are indicated by □, the hydrophilic sequences.

FIG. Figure 9 shows the physical map of FSME insertion plasmids as well as detection of the 10 plasmid DNA fragments containing FSME-specific sequences. In A) the ethidium bromide stained agarose gel is seen with the DNA fragments and in B) the result of Southern Blot hybridization with an "FSME Riboprobe" is shown.

A) Lane 1 λ DNA cut with Hindlll as size marker 15 Lane 2 pSCll DNA cut with Pvul

Lane 3 pSCll-P6 DNA cut with Pvul Lane 4 pSCll-P6 DNA cut with BamHI Lane 5 pSCll DNA cut with BamHI Lane 6 pSCll-F41 DNA cut with BamHI 20 Lane 7 pSCll-F41 DNA cut with EcoRI

Lane 8 pSCl 1 DNA cut with EcoRI

B) Following hybridization, fragments containing FSME sequences were detected at 2.585 kb in lane 3 0.919 kb in lane 4 1.019 kb in lane 6 1.879 kb in lane 7

These fragments are expected from the cloning (see Fig. 7). DNA in lanes 1, 2, 5 and 8 does not hybridize as it is only lambda DNA or pure pSC11 plasmid DNA.

In DK 175515 B1

I I

FIG. 10 shows a Slot Blot hybridization for detection of FSME vaccinia virus recom

recombinants. Uninfected cells, cells infected with the virus recombinant I

H vSC8 (Dr. Moss), and cells infected with 4 single isolates from virus recombination

nante P6, was transferred to nitrocellulose filter and hybridized with 32 P-labeled I

In 5 pSC11 DNA (nick-repair) in A or with an FSME-specific "Riboprobe" in B.

no signals were found with cells that were not infected. The recombinant virus I

vSC8 hybridizes with pSC11 due to the TK sequences. The FSME-specific I

However, as expected, "Riboprobe" did not produce a positive signal with vSC8. Cells, I

infected with single isolates of the viral recombinant P6 (1-4), exhibit I

10 hybridization both with the "Riboprobe" (due to the FSME sequence) and with I

H pSC11 DNA (due to the TK sequence). IN

FIG. 11 shows DNA fragment patterns obtained after cutting with HindIII from I

wild-type virus virion DNA and from vaccinia virus FSME recombinants. It can be seen that during the recombination process, the HindIII J fragment disappears in the cutting medium.

In the pattern of DNA from the virus recombinants. The original HindIII J fragment I

I with a size of approx. 5 kb occurs only in lane 6 of the agarose gel. All other DNA preparations do not contain the HindIII J fragment.

In 20 Lane 1 λ DNA cut with BstEII as the length standard I Lane 2 DNA from the FSME recombinant F41 (insertion plasmid pSCll-F41) In Lane 3 DNA from the FSME recombinant P6 (insertion plasmid pSC11-P6)

Lane 4 DNA from the virus recombinant PI (insertion plasmid pSC11)

Lane 5 DNA from the viral recombinant vSC8 25 Lane 6 DNA from virions from the wild-type strain WR.

In FIG. 12 shows a detection of FSME-specific sequences in HindIII DNA fragments I from virus recombinants by hybridization with a 32P-labeled FSME "Riboprobe" (A). As a control, the same filter was hybridized again with a 32P (nickel | I 30 repair) labeled pSC11 plasmid DNA (B).

In A) The FSME-specific "Riboprobe" hybridizes exclusively with DNA

I from the virus recombinant F41 in lanes 1, 2 and 3 and with DNA from the recombinant P6 in lane 7. Neither hybridization can be ascertained with the recombinant PI in lanes 4, 5 and 6 or with DNA from the recombinant vSC8 in lane 8 and DNA from uninfected cells in lane 9.

B) The radiolabeled pSC11 DNA hybridizes to all DNA preparations detected with the FSME-specific "Riboprobe". This can be explained by the fact that pSC11 DNA has the vaccinia TK sequences necessary for the recombination. However, for this reason, this pSC11 plasmid DNA also hybridizes with DNA from recombinant PI in lanes 4, 5 and 6 as well as with DNA from recombinant vSC8 in lane 8. DNA from uninfected 10 cells does not hybridize (lane 9).

FIG. Figure 13 shows a Western Blot analysis of FSME-specific protein in extracts from Vero cells infected with the viral recombinant P6. The proteins were separated in a 15% polyacrylamide gel and prepared for Western Blot by standard methods.

As a control, the proteins from these infected cells were incubated with a negative human serum (lanes 1 and 3). With a positive human serum containing antibodies to FSME virus, the expected FSME-specific antigen with a size of approx. 38,000 D in lanes 2 and 4. The signals in lanes 1 and 3 obtained with the negative serum appear to be nonspecific.

The DNA of the present invention is prepared and the sequence determined by the following general procedure: First, viral RNA is extracted from the western subtype of FSME virus or from 25 cells infected with the western subtype of FSME virus. Using this RNA as a matrix, a double-stranded cDNA is synthesized, e.g., using reverse transcriptase. By recombinant DNA techniques, this cDNA is inserted into a vector DNA such as Escherichia coli plasmid DNA to obtain a recombinant plasmid. Recombinant plasmids are used to transform suitable host cells, such as, for example, E. coli strain HB101, for amplification of the plasmids or for expression of the corresponding proteins. Single colonies with insert-containing plasmids are identified by the plasmid mini-preparation method of Birnboim and Doly (Nucleic Acids Research 7, 1195-1204, 1979). The base sequence of the DNA specific for the western subtype of the virus found in the recombinant

I DK 175515 B1 I

I I

In plasmid, is determined by a rapid DNA sequencing method such as by dideoxy-I

In the chain termination method. IN

I A detailed description of the method for producing cDNA, recombinant I

In 5 plasmids, cells transformed with these plasmids, and determination of I

In the nucleotide sequence is as follows: first, viral RNA from the western subtype I

I of FSME virus is obtained from purified virus. To this end, the western subtype I

of FSME virus is grown in primary chicken fetal cells, concentrated by ultracentrifuge.

and purified by two cycles of sucrose density gradient centrifugation. IN

10 After solubilization of the proteins with SDS in the presence of proteinase K and I

For example, in RNAse inhibitor, for example, by incubation for 1 hour at 37 ° C, the RNA is extracted with

phenol and precipitated with ethanol. Using this RNA as matrix I, the complementary DNA of the viral RNA is then synthesized in vitro by reverse transcriptase (e.g., from the "Avian Myeloblastosis" virus) by the method of Gubier and Hofmann (Gene 25, 263-269, 1983) Using primers such as hexanucleotide primers derived from calf thymus DNA are incubated

In the viral RNA under suitable conditions, eg as described in the manual "cDNA

Synthesis System "from Amersham (England), with a reverse transcriptase together with deoxyadenosine triphosphate (dATP), desoxythymidine triphosphate (dTTP), I deoxyguanosine triphosphate (dGTP) and desoxycytidine triphosphate (dCTP) as

In streets. The cDNA.RNA hybrid thus obtained is then treated with RNAse H

under certain conditions under which "nicks" are produced in the RNA from

In the bridging molecule. Then, E. coli DNA polymerase I can be used to replace I

The RNA strand effectively, with the nick-treated RNA serving as a primer. The thus-obtained double-stranded DNA (dsDNA) is deproteinized by phenol-I chloroform extraction and precipitated with ethanol, and residual RNA fragments I are removed by treatment with RNAse (e.g., in TE buffer, 30 minutes at 37 ° C).

Cloning of the dsDNA is performed, for example, using synthetic adapters. For this

For purposes, the dsDNA is treated with the Klenow fragment of E. coli DNA polymerase I

under appropriate conditions (Maniatis et al., Molecular Cloning, CSH 1982) I to provide a maximum number of clumpable ends that can be cloned, followed by I phenol extractions for the deproteinization. The blunt-ended dsDNA is phosphorylated at the 5 'end by polynucleotide kinase according to standard methods (Maniatis et al., In Molecular Cloning, 1982). The phosphorylated dsDNA is incubated under appropriate conditions with Bam HI-SmaI adapters in the presence of DNA ligase. The reaction mixture is placed on a 1% agarose gel and separated, and various size ranges of cDNA associated with adapters are cut out of the gel and purified, for example by electroelution. The DNA thus obtained is phosphorylated on rI5 with polynucleotide kinase as described above. On the other hand, the plasmid DNA to be used as a vector DNA, for example, E. coli plasmid pBR322 DNA, is cut with the restriction endonuclease BamHI and dephosphorylated using bacterial alkaline phosphatase. The synthetic dsDNA containing BamHI adapters at both ends and the BamHI cut vector DNA are incubated under appropriate conditions to allow hybridization of complementary sequences at the ends of both DNA molecules and ligated with T4 DNA ligase.

The recombinant DNA molecule is then used to transform a competent E. coli strain (e.g., E. coli HB101) as described by Maniatis et al.

(Molecular Cloning, CSH, 1982). The vector DNA used contains two genes that confer resistance to ampicillin and tetracydine, respectively. By the cloning method used, the tetracydine resistance gene is destroyed. For selection of recombinants likely to contain virus-specific insertions, the transformed bacteria are grown in the presence of ampicillin and then analyzed for tetracycline sensitivity. Plasmids from tetracycline-sensitive colonies are isolated by the plasmidine preparation method (Birnboim and Doly, Nucleic Acids Research 7, 1195-1204, 1979), and the size of the insertions is analyzed by restriction enzyme analysis using SmaI and separation of the obtained fragments on 1% agarose gels. 1 2 3 4 5 6 7 8 9 10 11 ___ _ - --—— -— ^^

The base sequence of these insertions is determined by the dideoxy chain termination method 2 of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977). The won 3 sequence is examined for homologies with already known sequences of other 4 flaviviruses (Rice et al., Science 229, 726-733, 1985; Dalgarno et al., J. Mol. Biol. 187, 5,309-323, 1986; Castle et al., Virology 147, 227-236, 1985; Castle et al., Virology 6 149, 10-26, 1986; Wengler et al., Virology 147, 264-274, 1985; Pletnev et al., 7 FEBS 3660 200, No. 2, 317-321, 1986) using computer programs to locate the examined sequence in the overall sequence of the genomic RNA.

9

FIG. 1 shows the complete nucleotide sequence and FIG. 3 shows the corresponding amino acid sequence of the structural region of the western subtype of the FSME virus genome.

I DK 175515 B1 I

I 16 I

I The exact amino terminals of E and M are determined by N-terminal amino acid I

In sequence analyzes, for example, using the manual method that is I

In described by Chang et al. (FEBS Letters 93, 205-214, 1978). For this purpose, iso- I

In 5, the E protein, for example by preparative SDS-PAGE, is electroeluted from the gel and sub-eluted.

You throw in an N-terminal sequence analysis. The resulting sequence is Ser-Arg-Cys, I

In and therefore the amino terminal of E is in exactly the same position as other I

In flaviviras, ie. two amino acids before the first well-preserved cysteine residue (Fig. 3). IN

I The following method can be used to determine the amino terminal of the M protein. IN

Purified virus is solubilized with a nonionic detergent (e.g., Triton X 100) and pro-I

In complexes containing E and M, density gradient centrifuge is recovered

Low in a detergent-free sucrose gradient (Heinz and Kunz, J. Gen. Virol. 49, 125-1

I 132, 1980). Sequence analysis of these protein complexes leads to the following double I

In sequence: I

In Ser-Arg-Cys-I

In Ser-Val-Leu- I

wherein Ser-Arg-Cys corresponds to the sequence of E, and Ser-Val-Leu is the amino term I

20 needles end of the M protein. These terminals are indicated in the FIGS. 1 and 3

You see. IN

Thus, for the first time, the present invention provides nucleotide sequence I

the late 5 'region of the western subtype of the FSME virus genome, including those I

In 25 sequences encoding structural proteins, and as a result, it provides I

in addition, the amino acid sequences of these structural proteins. IN

In the base sequence shown in FIG. 1 is a preferred example of a DNA encoding pol I

In lypeptides with the properties of the structural proteins of the western subtype of I

In 30 FSME viruses. According to the degeneracy of the genetic code, the same amino- I

Acid sequence is also encoded by base triplets other than those shown in the figure. IN

In the present invention, sequences altered by mutation also relate to I

In transposition and degradation, but which nevertheless encode an amino I

DK 175515 B1 17 acid sequence which still exhibits essential antigenic characteristics for the structural proteins.

Furthermore, the present invention not only relates to exactly the same 5 amino acid sequence as that shown in FIG. 3, which is just one example of a sequence of a natural isolate of the western subtype of FSME virus. It is a well-known fact that the genomes of RNA viruses are subject to higher mutation rates than those found for DNA viruses or cellular genes, due to the greater frequency of RNA polymerases and the lack of a correction mechanism (Netherlands). et al., Science 215, 1577-1585, 1982; Reanney, Ann.

Microbiol. 36, 47-73, 1982). Depending on the characteristics of the virus, these mutations can lead to the rapid development of a new virus type due to antigen drift, as is the case with influenza viruses (Both et al. In "The Origin of Pandemic Influenza Viruses" , Elsevier, 1983). On the other hand, 15 RNA viruses may be more stable with respect to their antigenic structure under natural ecological conditions, presumably due to functional coercive mechanisms that do not allow for far-reaching structural changes in antigen-active proteins.

Nevertheless, some degree of variation must be taken into account, and this can be demonstrated by sequence comparisons between different natural isolates.

20

This can be done, for example, by sequencing RNA from different isolates using the dideoxy chain termination method with synthetic oligonucleotides as primers prepared according to the 1 shows the sequence of the prototype isolate. 1

Analysis of the nucleotide sequences of such different isolates corresponding to the E protein has shown diverse nucleotides. The following nucleotide replacements were found in RNA from a natural isolate (ZZ-9) in comparison with Neudorfi:

I DK 175515 B1 I

I 18 I

In Position of FSME ZZ-9 Amino Acid I

In the nucleotide replacement I

I 2375. A U Thr - Ser I

I 23A7 U C I

I 2323 AG I

I 2317 AG I

I 2281 C U I

I 2266 U C I

I 2263 AG I

I 2021 C U - I

I 1876 C U I

I 1868 A G Met - Val

I 1773 C U Ala - Val I

I 1668 A G Glu - Gly I

I 1663 C U I

I 1661 A G Asn - Asp I

I 1660 C U I

I 1 A72 U C I

I 1451 A G Ile - Val I

I 1 3A8 C U I

I 1 11A u C I

I 1111 u c I

I 1090 C U I

I 1 0 A 8 u c I

I 99A U C I

DK 175515 B1 19

The observed replacements are an expression of the natural degree of variation of the FSME virus that does not lead to a new serotype. Sequence homologies between the E proteins of flaviviruses that do not belong to the same serocomplex are in the range 40-50%, e.g., 44.4% homology between YF and MVE virus, and in the range 70-80% among members. of the same flavivirus serocomplex, e.g., 77.1% homology between WN and MVE virus.

A comparison between the published nucleotide sequences of the far eastern subtype (Pletnev et al., FEBS 3660 200, no. 2, 317-321, 1986) and the western subtype showed a homology of about 85%.

All sequences exhibiting small variations in relation to the prototype sequences, as they appear in other natural isolates of the western subtype of FSME virus, are encompassed by the present invention. Moreover, such variations can be obtained by in vitro modification of the nucleic acids. Therefore, the invention encompasses all sequences that hybridize under stringent conditions, e.g., at least 90% nucleotide sequence homology to the provided sequences or portions of sequences that preferably encode proteins or peptides having the essential characteristics of antigenic determinants of structural proteins of the western subtype of FSME virus.

It is known to those skilled in the art to insert the provided DNA or portions thereof into suitable vectors and to use the recombinant vectors for transformation of suitable cells, either to amplify the DNA or to obtain expression of the corresponding proteins. Suitable hosts, vectors and conditions for these operations are well known to those skilled in the art. There are many publications describing the synthesis of foreign proteins by recombinant DNA technology (an overview is given, for example, in "Maximizing Gene Expression" (Reznikoff and Gold, ed.), Butterworths, 1986;

Harris, Gen. Meadow. 4, 127-183 (Williamson, ed.), Academic Press, 1983; Wetzel and 30 Goeddel, The Peptides 5, 1-64, 1983). By these methods, proteins encoded by cloned DNA can be expressed either in bacteria, in yeast or in mammalian cells. Furthermore, it is well known to use such expressed proteins as vaccines for immunization purposes (Valenzuela et al., Nature 298, 347-350, 1982; McAleer et al., Nature 307, 178-180, 1984) or as diagnostic antigens.

I DK 175515 B1 I

I 20 I

In foreign DNA or RNA sequences can also be introduced into the genomes of living I

In viruses, forming recombinant viruses which can be used as living I

In Vaccine (review article by Mackett and Smith, J. Gen. Virol. 67, 2067-2082, 1986). IN

I I

Likewise, a combination of different genes derived, for example, from different I

In viruses, simultaneously expressed by recombinant DNA technology and used for vaccine I

tion (Perkus et al., Science 229, 981-984, 1985). The invention thus encompasses all of the following

combinations of sequences of the sequences provided with other sequences I

I 10, such as, for example, genes encoding other proteins, or sequences that contribute to I

In expression of the proteins, such as, for example, promoters, enhancers, polyadenylation I

In or splice signals. IN

It is known to those skilled in the art that one does not necessarily have to use all of them naturally

In proteins present for immunization or diagnostic purposes (Lerner, I

In Nature 299, 592-596, 1982; Arnon, TIBS 11, 521-524, 1986). In the case of the I

For example, in the western subtype of the FSME virus E protein, it has been shown that

Seration with a proteolytic fragment of 9000 daltons can be induced by hemagglutinase I

In anti-ionizing and neutralizing antibodies, which fragment also reacts

In 20 with polyclonal immune sera obtained by immunization with all natural I

In straight protein (Heinz et al., J. Gen. Virol. 65, 1921-1929, 1984). Other proteolytic I

In fragments may also be suitable for immunization or diagnostic purposes. IN

Proteins or parts of proteins used as vaccines or diagnostics

25 agents can not only be prepared by recombinant DNA technologies as described in I

In the above, however, the sequence information I provided by the present invention

You can also form the basis for the chemical synthesis of oligopeptides. Within this

In the area there is much literature: synthesized peptides have been produced

In accordance with DNA sequences encoding many different proteins, these are I

I have been used for a variety of purposes such as, for example, molecular biology and immunology

In icy studies (Lerner et al., Cell 23, 309-310, 1981; Lerner, Nature 299, 592-1

i

In 596, 1982) or vaccinations (Shinnick et al., Ann. Rev. Microbiol. 37, 425-446, I

In 1983; DiMarchi et al., Science 232, 639-641, 1986). Preparation and Use of I

In peptides or combinations of peptides similar to those of the present I

Sequences provided in the invention, therefore, form part of the prior art. IN

DK 175515 B1 21

In addition, it is known to those skilled in the art to use the nucleic acids and sequences provided by the present invention for the production of hybridization probes, e.g., for the determination of viral RNA in forest rafts or body fluids (Meinkoth and Wahl, S Anal. Biochem. 138, 267 -284, 1984; Kulski and Norval, Arch. Virol. 83, 3-15, 1985).

These can be produced either by recombinant DNA technologies or by chemical synthesis of oligonucleotides corresponding to the sequences provided.

The invention is illustrated by the following examples.

EXAMPLE 1

Propagation and Purification of FSME Virus .15

A 10% suspension of mouse brains infected with the western subtype of FSME virus was used to infect primary chicken embryonic single-layer cultures maintained with 15 mM HEPES and 15 mM EPPS at pH 7.6 buffered minimal medium (MEM). After 40 hours of incubation at 37 ° C, the residue was clarified for 30 minutes 20 at 4 ° C and 10,000 xg, and the virus was pelleted by 3 hours ultracentrifugation at 4 ° C and 50,000 xg. The virus was then resuspended in an appropriate volume of TAN. buffer (0.05 M triethanolamine, 0.1 M NaCl, pH 8.0) and subjected to a zonal (rate-zonal) centrifugation in a 5-20% (w / w) sucrose density gradient at 170,000 xg for 110 minutes at 4 ° C. The virus peak was located by scanning the gradient at 254 nm and was subjected to an equilibrium density gradient centrifugation in a 20-50% (w / w) sucrose gradient for 18 hours at 4 ° C and 150,000 x g. dialyzed against TAN buffer, pH 8.0, to remove excess sucrose.

EXAMPLE 2

Preparation of Viral RNA

100 µg of purified virus was diluted in 400 μΙ of a proteinase K reaction buffer 35 (10 M Tris, pH 7.8, 5 mM EDTA, 0.5% w / v SDS). Proteinase K was added to a I DK 175515 B1

I 22 I

At final concentration of 200 µg / ml and the mixture was incubated for 1 hour at 37 ° C. IN

Then, the solution was deproteinized by twice extraction with the same I

volume (400 μΙ) of phenol and one-time extraction with chloroform / isoamyl alcohol I

(24: 1). Then, 26 μΙ of a 3 M sodium acetate solution was added and the RNA I

5 was precipitated with 2.5 volumes of ethanol. Prior to its further use, an aliquot I

of the RNA denatured with glyoxal and separated on a 1% agarose gel to control I

clay yield and quality of preparation. IN

In Example 3 I 10

Synthesis of double-stranded cDNA

5 µg of ethanol-precipitated RNA was resuspended in 40 µg of a single-stranded synthesis buffer (Amersham, GB) containing 5 µg of a "Random" oligonucleotide primer, heated for 1 minute to 70 ° C and then slowly cooled to room temperature. All Four H desoxynucleotide triphosphates were added to a final concentration of 1 mM.

In addition, 5 units of human placental ribonuclease inhibitor and 10 μΟ α-32Ρ dCTP were added to the mixture. Single strand synthesis was initiated by the addition of 100 units of reverse transcriptase (Amersham) and performed for 2 hours at 42 ° C.

Then, the reaction mixture was placed on ice and the reagents for the double strand synthesis were added in the following order: 93.5 μΙ of a double strand synthesis buffer I (Amersham, GB), 4 units of ribonuclease H, 23 units of E. coli DNA polymerase and water to a final volume of 250 μΙ. The double strand synthesis was performed by successive incubations at 12 ° C and 22 ° C (every 2 hours) and stopped at

For 20 minutes, warm the solution at 70 ° C. The double-stranded cDNA

You were incubated with 10 units of T4 DNA polymerase for 30 minutes at 37 ° C to obtain blunt ends. Finally, the cDNA was purified by phenol and chloroform extractions and precipitated with 2 volumes of ethanol.

EXAMPLE 4 I Adapter ligation

The cDNA was treated with polynucleotide kinase in the presence of 2 mM ATP

I 35 (Maniatis et al., Molecular Cloning, CSH 1982) to ensure that all 5 'ends were phosphorylated and thus could be ligated. BamHI-SmaI adapters were provided by Pharmacia. These adapters had an S 'protruding, with BamHI compatible protruding (sticky) end and a blunt end, and they contained the complete Narrow Recognition site within their sequence. First, only the blunt end had a 5 'phosphate group and could be ligated, while the protruding end was dephosphorylated. 5 µg of these adapters were mixed with the cDNA in 20 μΙ of the ligation buffer (50 mM Tris, pH 7.5, 5 mM MgC 2, 5 mM DTT, 1 mM ATP). The reaction was catalyzed with T4 DNA ligase (New England Biolabs) in an overnight reaction at 14 ° C.

EXAMPLE 5

Size separation of the cDNA The cDNA was separated on a 1% low melting agarose gel. Various size classes were excised from the gel and the DNA was extracted from standard phenol agarose by standard methods. Despite the small amount of available DNA which could hardly be detected by staining with ethidium bromide, the extraction process could be easily followed by the radioactive labeling incorporated into the cDNA.

The size separation step enabled selective cloning of large cDNA fragments and further served to completely separate the cDNA from non-ligated adapter molecules.

EXAMPLE 6 25

Cloning into a bacterial plasmid

The BamHI-compatible ends of the cDNA from different size classes were phosphorylated with polynucleotide kinase and deproteinization was performed by 30 phenol and chloroform extractions. The DNA was then precipitated with 2 volumes of ethanol and resuspended in a small volume of water. 20-30 ng of the cDNA was mixed with 100 ng of the Escherichia co // plasmid pBR322, which had been linearized and dephosphorylated by BamHI cutting to prevent self-ligation. These two components were ligated with T4 DNA ligase in 5 μΙ of the ligation buffer (see above) in a 35 overnight reaction at 16 ° C. The next day, the ligation mixture was diluted five

I DK 175515 B1 I

I 24 I

In water passages and then used directly to transform E. coli HB101-I

In cells. Competent bacteria were supplied by BRL (Maryland, USA) and transformed I

In with 5 μΙ of the diluted ligation mixture as recommended by the manufacturer. After I

Plating on ampicillin-containing agar plates typically yielded several hundred I

In 5 transformed cells. IN

In Example 7. IN

Identification of insert-containing plasmids I

I 10 I

In Single ampicillin-resistant colonies, were coated on tetracycline-containing agarplate.

In there. For approximately 5% of the colonies tested, sensitivity to I was established

In tetracycline. These were used to inoculate 2 ml of ampicillin-containing LB media I

and incubated overnight at 37 ° C in a shaker at 220 rpm. The I

For the next day, a plasmid rapid preparation was performed according to standard method I

I (Birnboim and Doly, Nucleic Acids Research 7, 1195-1204, 1979). The plasmids became I

In the cut with the restriction enzyme Smal and analyzed on 1% agarose gels (Fig. 4).

In this way, 21 plasmids containing insertions in the size range 2000-3500 bp were identified.

EXAMPLE 8 I Preliminary characterization of the insertions I 25 Insert-containing plasmids were prepared on a large scale by a standard method (Maniatis et al., Molecular Cloning, CSH, 1982) and purified by ethidium bromide I density gradient centrifugation. Ca. 500 ng of the plasmid DNAs were cut with a variety of restriction enzymes and analyzed on 1% agarose gels. Thus, characteristic band patterns for each plasmid were obtained, which allowed an initial prediction of which insertions could contain overlapping or different sequence regions.

EXAMPLE 9

Sequence analysis of the insertions in clone A5 5 One of the insert-containing plasmids designated plasmid A5 was cut with SmaI and separated on a 0.7% agarose gel. The band representing the entire insert was cut out of the gel and the DNA was purified from the agarose by electroelution. The isolated fragment was cut by various reactions with frequently intersecting restriction enzymes forming blunt ends such as, for example, Rsal, Alul 10 and Haelll. The double-stranded form of the bacteriophage M13mp8 was cut with SmaI and dephosphorylated. The mixtures of DNA fragments were deproteinized, precipitated and ligated with the M13 vector as described above. Transformation of E. coli JM105 cells, identification of recombinant phages by the color selection method, and preparation of single-stranded DNA was performed by standard methods. Single clones were sequenced by the dideoxy sequencing method described by Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977). As the primer, the commercially available M13 universal primer was commonly used. Thus, the sequence of a large number of different fragments of the clone A5 was determined, ultimately leading to the overall DNA sequence of this clone. By comparing this sequence with published genome sequences of different flaviviruses, it was determined that A5 contains the region of the viral genome encoding the structural proteins C, M and E with the exception of the amino-terminal half of the C protein. The exact location of A5 is shown in FIG. First

EXAMPLE 10 cDNA Synthesis Using an FSME Virus Specific Primer An FSME virus specific primer was used to construct a cDNA clone containing the entire 5 'region of the viral genome, including the complete sequence information on C protein, as well as most of the untranslated 5 'leader region. This oligonucleotide primer consisted of 14 nucleotides complementary to a region of the FSME genome encoding the C-terminal amino acids of the E-35 protein. Preparation of viral RNA and synthesis of double-stranded cDNA was performed.

I DK 175515 B1 I

I 26 I

conducted essentially as described above with the exception that this I

once only 1 µg of RNA and 0.5 µg of the synthetic oligonucleotide primer PI were used. IN

Finally, the incubation times for the double strand synthesis and T4 DNA were I

H the polymerase treatment reduced to ten! 1 hour and 3 minutes respectively. IN

I EXAMPLE 11

Cloning in the bluescript vector using EcoRI linkers 10 Using the information available at this time in the research, it could be assumed that the entire structural area contained no

Eco RI recognition sites. Therefore, synthetic EcoRI (New England Biolabs) linkers could be used for the cloning process. Ca. 1 µg of cDNA was ligated with 1 µg of linker DNA for 6 hours at 14 ° C using T4 DNA ligase and a buffer - 15 system as described above. After purification of the mixture by phenol and chloroform extractions and ethanol precipitation, the DNA was resuspended in 40 µl of a "medium salt" restriction enzyme buffer (CSH manual) and cut for 3 hours with an excess amount of Eco RI (80 units) at 37 ° C. . Then the mixture was separated on a 1% agarose gel and cDNA in the size range 1500-2500 bp was recovered from the gel by electroelution.

At the same time, the new versatile vector Bluescript KS M13 + (Stratagene) was cut with I EcoRI. 100 ng of this linearized plasmid was mixed with the entire cDNA preparation and the DNAs were coprecipitated with ethanol. Plasmid cDNA ligation I 25 was performed as described above. Ligation mixtures were gradually diluted from two I to one hundred times and used to transform E. coli XL-1 blue. Transformation and identification of insert-containing plasmids by color selection were performed exactly according to the manufacturer's instructions (Stratagene). Thus, 4 plasmids containing inserts with a length of approx. 2500 bp.

EXAMPLE 12

Sequencing of the clone P1-1 One of the insert containing plasmids designated P1-1 was used to produce single stranded matrix DNA. This was done by adding helper phage R408 to an exponentially growing bacterial culture containing the plasmid Pl-1 in an M.O.I. at 20: 1. After 8 hours incubation at 37 ° C in a shaker at 240 rpm, the supernatant containing phage particles was isolated and 10 single-stranded P1-1 DNA was prepared by standard methods. Different from the classical M13 system, the Bluescript system allows the production of single-stranded matrix DNA containing the entire cDNA sequence, thus rendering the time-consuming subcloning process redundant. Sequencing of P1-1 was performed by the standard videoxymy method using both universal M13-15 and synthetic oligonucleotide primers corresponding to different viral sequences such as primers. It was found that Pl-1 encompasses the entire region encoding structural proteins and extends 120 bp beyond the C protein start codon. The exact location of P1 is also shown in FIG. First

EXAMPLE 13

Sequencing of various natural isolates by direct sequencing of the RNA 1 2 3 4 5 6 7 8 9 10 11

The sequences encoding the structural proteins of various natural isolates of 2 the western subtype of FSME virus were performed by direct sequencing of 3 genomic RNAs with a modification of Sanger's dideoxy method, essentially described by Zimmern and Kaesberg (Proc. Natl. Acad. Sci. USA 75, 5 4257-4261, 1978). Different synthetic oligonucleotides corresponding to different 6 sites in the genome were used as primers. Ca. 1 µg of genomic RNA and 0.5 µg of 7 the respective primer were mixed in a volume of 10 µΙ, heated for 3 minutes 8 at 65 ° C and then slowly cooled to 42 ° C. The sequencing reaction was kata 9 lysed with reverse transcriptase (Amersham) for 15 minutes at 42 ° C. As a control, the FSME virus prototype Neudorfl was simultaneously sequenced and analyzed on 11 the same polyacrylamide gel. This enabled a simple and unambiguous identification

DK 175515 B1 I

ring of sequence variations in various natural isolates. Sequence deviations became I

found in positions described in the general section of the specification. IN

EXAMPLE 14 I

Amino-terminal sequence analysis of viral proteins I

1 mg of purified Western subtype of FSME virus was subjected to SDS-PAGE according to I

Laemmli and Favre (J. Mol. Biol. 80, 575-599, 1973) on 10% acrylamide gels. E- I

The protein was localized by staining a filter paper blot of the gel with ami

dose and the protein was excised from the gel and electroeluted using H

an ISCO elution apparatus for 5 hours at 3 W. The N-terminal sequence analysis I

was performed by that of Chang et al. described procedure (FEBS Letters

93, 205-214, 1978). The sequence obtained was Ser-Arg-Cys and thus enabled H

15 to determine the exact position of the E protein in the embodiment of FIG. 3. IN

Alternatively, protein complexes containing both E and M were prepared at I

solubilization of purified Western subtype FSME viruses with Triton X 100, I

followed by density gradient centrifugation in detergent-free density gradients such as I

20 described by Heinz and Kunz (3rd Gen. Virol. 49, 125-132, 1980). As expected, you gave

the amino-terminal sequence analysis a double sequence, namely H

Ser-Arg-Cys I

Ser-Val-Leu. IN

25 I

Since it was already known that Ser-Arg-Cys was derived from the E protein, the sequence I

late Ser-Val-Leu thus identified as M's amino terminal (Fig. 3). IN

EXAMPLE 15 29 DK 175515 B1

Preparation of insertion plasmids containing foreign genes by homologous recombination in the vaccinia HindIII J fragment by cloning 5 of 5 'end sequences of the cloned cDNA from FSME virus. 5 is a plasmid A5 shown in known manner as a pBR322-derived plasmid. As shown in FIG. 5, plasmid A5 contains the DNA sequence encoding the E protein of FSME virus. In FIG. 5, restriction endonuclease cutting sites of selected sub-fragments are also shown, the regions coding for FSME E protein encoding. The arrow shows the direction of transcription in the genomic RNA.

Additional viral and plasmid material, as well as the methods used to produce plasmids containing specific DNA sections encoding the E-15 protein, are described below: 15.1. Virus and cell culture

A vaccinia virus wild-type strain WR is available in the ATCC 20 landfill number VR-119. Human TK'143 cells which do not produce thymidine kinase are widely available and are deposited in the Institut Pasteur (Accession number 1-732). Vaccinia virus is propagated in Verocells approx. between the passages 47 and 55. The cells are grown in MEM (minimal essential medium) with 5% FCS (fetal calf serum), antibiotics and glutamine. The virus strain to be used for the re-combination is passed once through HAT medium (hypoxanthine, aminopterin, thymidine) containing 5% FCS to select a TK + wild-type virus. Recombinant TK virus is determined on human TK'143 cells grown in MEM with 5% FCS containing 25 pg / ml BUdR. The recombinants can be found by overlaying the cell single layer with low-melting agarose containing MEM, 5% FCS, 25 μς / ιηΙ BUdR 30 and 250 ng / ml X-gal. The cell culture is kept for 48-72 hours in an incubator at 37 ° C. The amount of spontaneous TK 'virus mutants is determined by infection of single TK "cell layers in the presence or absence of 25 Mg / ml BUdR. Usually, one mutant can be observed among 104 viruses (Mackett et al. In" DNA cloning Vol. II, and a practical approach ", IRL Press, DM Glover (ed., 1985).

35

I DK 175515 B1 I

I 30 I

I 15.2. Plasmids and cloning methods

In the plasmid A5 shown in FIG. 5, and which is a pBR322-derived plasmid, has the I shown

In restriction endonuclease map. IN

I. IN

In FIG. 6 shows the plasmid pSC11, which contains the E. coli lacZ gene for "blue color" - I

In determination, the TK flanking sequences and a vaccinia promoter. Plasmid I

The pSCll is well known and widely available and can be obtained especially from the National Institute of I

In Health. IN

I 10 I

In all the restriction endonucleases used for the modification of the DNA, I

You were used according to the manufacturer's instructions for use. Southern Blot Analysis and I

In isolation of plasmid DNA was performed by standard methods (Maniatis et al., I

In Molecular Cloning, CSH 1982). IN

I 15 I

Radioactively labeled probes were either obtained by nick-repair methods or by I

In the pSP6 "Riboprobe" method (Green et al., Cell 32, 681-694, 1983). IN

In the "Riboprobe" used in the present invention, I was prepared

I 20 by ligation of an internal FSME nucleotide sequence of ca. 1.0 kb in plasmid pSP64. IN

Before starting RNA synthesis, the plasmid was linearized with EcoRI. A typical I

The assay consisted of: I

In Tris-HCl pH 7.5 40 mM I

I MgCl2 6 rnM I

I 25 Spermidine 2 mM I

In NaCl 20 mM I

In DTT 10 mM I

In ATP, CTP, GTP every 0.5 mM I

In Human placental RNAse inhibitor 20 U I

I 30 pSP64-P5 X EcoRI 2 Mg I

In o32P UTP (400 Ci / mmol) 100 µCi I

In SP6 RNA polymerase 4 U I

Up to a total volume of 20 μΐ I

In 35 Incubation: 1 hour at 40 ° C. IN

DK 175515 B1 31

The nucleotides that were not incorporated were separated from the radiolabeled cRNA by column chromatography.

15.3. In vivo recombination assay

Infection and transfection:

Recombinations were performed essentially as described by Mackett 10 et al. (see 15.1). In addition to human TK 'cells, Vero cells or chicken embryo fibroblast cells could also be used as host cells. Ca. 1-3 hours before transfection, cells were infected with a multiplicity of infection (m.o.i.) of 0.01-0.05 pfu / cell with a wild-type WR strain. HEPES buffered saline solution for DNA transfection was prepared as follows: 10 x HBS containing 8.18% NaCl (w / v), 5.94% HEPES (w / v) and 0.2% Na '/ zZHPOV (w / v) was diluted in 2 x HBS before use. The pH was adjusted to exactly 7.05 with 1N NaOH. The buffer was diluted with 1 x HBS and sterilized by filtration. To 1 ml of this 1 x HBS was added a solution of 1-5 µg of plasmid DNA and 10-20 µg of carrier DNA (either vaccinia virus wild type or salmon sperm DNA). In particular, the addition of the carrier DNA 20 appeared to be substantial. The stirred mixture was thoroughly mixed for 1 minute on a vortex mixer. Then, 2 M CaCl 3 solution was slowly added to a final concentration of 0.125 M. A DNA precipitate became visible after 20-30 minutes at 25 ° C. The medium was removed from the infected cells, the single layer was washed once with the cell culture medium, and after removal of all the remaining fluid, the DNA precipitate was slowly added. Thirty minutes later, fresh medium was added at 25 ° C and the cells incubated at 37 ° C. After 3-4 hours, the medium was again removed and replaced with fresh cell culture medium. The cells were usually harvested after 24 or 48 hours at scraping and sedimented at low speed, and the cell pellet was collected in a total volume of 500 μΙ medium.

30

Plaque assay and purification of recombinant viruses:

The virus suspension was frozen three times and freshly thawed; 60 μΙ of the suspension was incubated with 1/5 volume of 1.25% trypsin for 30 minutes at 35 ° C. A series of three dilutions (1:10) was added to a confluent single layer of TK '

I DK 175515 B1 I

I 32 I

In 143 cells grown in 10 cm 2 plates. After 30-60 minutes incubation at I

At 37 ° C, the medium was removed and then 3 ml of the medium was added 25 µg / ml bromine.

In deoxyuridine. After 24 or 48 hours at 37 ° C, the medium was again removed and replaced with I

I with an agarose overlay containing 25 pg / ml bromodeoxyuridine and 125 pg / ml I

In 5 X gal. After a further 24-48 hours of incubation, blue plaques could be detected. IN

In Some of these plaques were removed and suspended in 500 μΙ medium. This one

In virus-containing suspension, as described above, trypsin was treated. IN

In Plaque purifications were performed at least twice. IN

I 10 15.4. Preparation and characterization of both wild-type and recombinant I

In vaccinia DNA I

In Infected cells, scrap of 175 cm 2 flasks and washed twice with PBS-I

In Ca 2+ / Mg 2+ at a pH of 7.4. After centrifugation, the cells were resuspended I

In 15 in 250 μΙ PBS. The gels were frozen for a short time three times and thawed again. A volume I

I on 250 μΙ of a 2 x cell lysis buffer (1% Triton X 100; 70 mM β-mercaptoethanol; I

In 40 mM EDTA) was added and the cells were lysed for 5 min on ice. The core became you

separated from the cytoplasm for 1 minute at 16,000 x g. The residue containing I

In aggregated virus particles, transferred to a new tube and centrifuged for 30 minutes I

In 20 at 16,000 x g to pellet the virus. Then virus lysis buffer (10 mM Tris-I) was added

HCl, pH 8; 1 mM EDTA; 5 mM β-mercaptoethanol; 200 mM NaCl; 1% SDS; IN

150 µg / ml proteinase K) added to the pellet in an amount of 100 µΙ and incubated in I

For 30 minutes at 56 ° C. The nucleic acids were then extracted 3x with Tris-HCl-I

In saturated phenol and then re-extracted with chloroform / isoamyl alcohol (24: 1).

25 Care was taken to ensure that the DNA was not subjected to any shear forces. The nucleic acids were precipitated with 0.1 M NaCl and a 2.5-fold volume in ethanol at -20 ° C overnight. Approximately 10-20 µg of the DNA could be isolated from 107 cells. After centrifugation (30 minutes at 17,000 xg) ), the DNA was dissolved in a small volume of 20-40 μΙ H 2 O. The DNA was cut with the restriction endonuclease 30 HindIII and the fragments separated on 0.6% agarose gels in TA buffer (Rice et al., J. Virol. 56 , 227-239, 1985).

35 33 DK 175515 B1 15.5. Detection of nucleic acids by hybridization

Southern Blot 5 After transferring the DNA from the gels (30 ', 0.5N NaOH and 2x10', 10x SSC / 0.5M Tris-HCl pH 7.5) to nitrocellulose filters, they were baked for 4 hours at 80 ° C. The filters were prehybridized for 6 hours at 65 ° C in 5 x Denhardt solution (1 x Denhardt = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA). The wet filters were sealed in plastic bags and incubated for 18 hours at 43 ° C with the hybridization solution and the radioactive probe. The filters were washed at 65 ° C three times, each time 30 'with each time 2 x SSC, 1 x SSC and 0.1 x SSC with 0.1% SDS and 20 mM NaPO 4. After air-drying the filters, they were exposed to Kodak X-omat AR X-ray film.

A "Riboprobe" vector (pSP6) containing an FSME fragment from the one shown in FIG. 5, plasmid A5, was used to prepare an FSME virus specific "Riboprobe". Radioactively labeled cRNA was mixed with a hybridization solution (50% formamide, Amberlit purified; 5x SSC, 1x Denhardt solution, 0.1% SDS; 100 μ9 / ηηΙ herring sperm DNA) The total volume of the hybridization solution was determined, with a 50 μΙ. 100 μΙ / cm2 filter required for optimal hybridization conditions.

The amount of radioactive cRNA was approx. 107 cpm / ml or approx. 106 cpm / cm2 filter. The solution was heated for 5 minutes at 65 ° C.

Spot Blot 25 These experiments were performed according to Mackett et al (see 15.1.) With minor modifications. Material from 1/4 of the isolated plaques was used to infect a 35 mm Petri dish. 48 hours after infection, cells were harvested and washed in a buffer containing 50 mM Tris, pH 7.5, and 100 mM NaCl. Finally, the cells were resuspended in 500 μΙ of this buffer. 100 μΙ aliquots were vacuum-vacuumed on 30 nitrocellulose. After drying, the filters soaked in 0.5 M IMaOH for 3 minutes were placed on chromatography paper. Then, the filters were neutralized on paper filters soaked in 2 x SSC / 1 M Tris-HCl, pH 7.5 for 3 minutes. The filters were then baked for at least 2 hours and then treated in the same manner as described above for the Southern Blot hybridization. .

35 I DK 175515 B1

I I

I 15.6. Protein Analysis I

In Vero cells or TK'143 cells were infected with a m.o.i. of 1 pfu / cell in a total I

In volume of approx. 1-2 ml / 25 cm 2 flasks. In some experiments, the cells were labeled with I

In 5 100 μΟ 35S-methionine / ml. When CPE was strong, the cells were harvested, washed 2 times I

I in PBS and finally resuspended in 100 μΙ RIPA buffer (150 mM NaCl; 10 mM Tris-HCl, I

At pH 7.2; 1% sodium deoxycholate; 1% Triton X 100; 0.1% SDS; 100 µg / ml PMSF) I

I (50-150 μΙ for 5 x 105 - 1.5 x 106 cells). After 10 minutes on ice, the probes became ul

In the grating for 30 seconds. To 8 μΙ of this protein mixture, 12 μΙ of an I was added

In 2 x boiling buffer (0.2 M DTT; 4% SDS; 0.16 M Tris-HCl, pH 6.8; 20% glycerol; 1/100 L

In volume 5% BPB in ethanol); the proteins were denatured for 10 minutes at 100 ° C

And subjected to electrophoresis on 15% SDS-polyacrylamide gels (Bodemer et al., I

In Virology 103, 340-349, 1980). The proteins were transferred from the gel to nitrocellulose I

In filters in a buffer containing 0.192 M glycine, 0.025 M Tris, pH 8.3 and 20% I

In 15 methanol at a current of approx. 1.5 A for 2.5 hours. The nitrocellulose filters became I

Then incubated in a blocking solution "NET" (0.15 M NaCl, 5 mM EDTA, I

In 50 mM Tris, pH 7.4, 0.05% Triton X 100, 2% BSA). Human serum and an enzyme I

In the second labeled antibody directed against human immunoglobulins antibody was added in NET in an I

In dilution of 1: 500 and incubated for 2 hours. The tapes were washed with NET in 3 x 10 L

For 20 minutes and then soaked with TBS (20 mM Tris, pH 7.5, 0.9% NaCl, 2% I

In BSA) for 3 x 10 minutes. The bands were developed in 50 ml of TBS and the chromogenic I

In substrate DAB and H2 O2. IN

I 15.7. Preparation of insertion plasmids I

I 25 I

With the materials and methods described above, plasmids, I, were now prepared

In which contains the DNA sequences of the E protein. First, plasmid A5 (Fig. 5) was I

In the cut with restriction endonucleases PvuII, FnuDII and Haell. The fragments I

I was inserted into the unique Sma I site of plasmid pSC11 (Fig. 6). The three FSME- I

In 30 fragments were isolated from agarose gels by electroelution and ligated with I

In the plasmid pSC11, the latter also linearized in the unique Smal

Instead. The physical map of these plasmids is shown in FIG. 7. The three fragments have an H

In size of 1022 bp (PvuII), 1485 bp (FnuDII) and 1553 bp (Haell). The three mentioned

In restriction endonucleases were used because the nucleotide sequence encoding H

In the inner part of the E protein, both with and without hydrophobic I had been found

35, DK 175515 B1 signals or membrane anchoring sequences at a hydropathy plot performed with an IBI Pustell computer program (Fig. 8). The orientation of the inserted protein E sequences with respect to the p 7.5 E / L vaccinia promoter was determined by Southern Blot analysis. Cutting the pSC11-P6 plasmid with Pvul or BamHI clearly yielded a 2,585 kb or 0.919 kb fragment (Fig. 9A, lanes 3 and 4), both of which hybridized with a 32 P-labeled "Riboprobe" (Fig. 9B, lanes 3 and 4). The plasmid pSC11-F41 could be cut into fragments with BamHI or EcoRI; fragments with a size of 1.019 kb or 1.879 kb both hybridized exclusively with the "Riboprobe" (Fig. 9B, lanes 6 and 7).

1 ° 15.8. In vivo recombination with FSME insertion plasmids and wild-type vaccinia virus

Vero cell single layers that had been infected with the vaccinia strain WR were transfected with plasmid DNA. After 48-72 hours, a strong cytopathogenic effect became visible and the cells were harvested. The virus was isolated from the cells by repeated freezing and thawing as well as trypsin treatment as described. Then, TK 'cell monolayers were infected with this virus suspension. The cells grew in the presence of BUdR. Plaques in development were visualized during an agarose-20 overlay containing the X-gal chromogenic substrate. Virus dilutions were performed to give 10-30 plaques per 35 mm plates. Single blue plaques were removed and used for the further infection of TK 'cells for purposes of further plaque purification of the infection selection or of Vero cells for the analysis of viral macromolecules.

25 15.9. Spot Blot analysis to find recombinant viruses

Small probes (approximately 100 μΙ) of infected single cell layers from 35 mm plates or 25 cm 2 culture flasks were divided and hybridized with a nick-repair labeled pSC11-30 plasmid (which did not contain FSME sequences) or with the FSME "(fig.

10). Skin-infected cells showed no signal with both probes. The vSC8 virus, which contained the lacZ gene and TK vaccinia sequences but no FSME sequences, gave a negative result with the FSME "Riboprobe". However, as expected, all cells with viruses from four different individual plaque isolations were positive with the FSME-35 and the pSC11 probe.

I DK 175515 B1 I

I 36 I

I 15.10. Analysis of DNA from recombinant viruses I

In Vero cell monolayers were infected with a m.o.i. of 1-10 pfu / cell with plaque op

In 5 purified, propagated vaccinia recombinants. The virus DNA was isolated from virus I

In particles nucleated from infected cells and analyzed by restriction endo-I

In nuclease cutting as well as Southern Blot hybridization. The 5 kb HindIII J fragment

Into wild-type DNA (as shown in Fig. 11, lane 6) was replaced with a larger I

In HindIII fragment, which consisted of the original HindIII J fragment and the DNA I

In 10 sequences that had been inserted into the TK gene site by in v / Vo recombination. vSC8- I

The virus contained a 3.5 kb lacZ gene and the HindIII J fragment migrated to the

This is at a size of approx. 8.5 kb (fig. 11, lane 5). However, since larger fragments I

You could not easily be separated into low percentage gels, a final I was performed

In the determination of the recombinant virus DNA by Southern Blot hybridization (Fig. I

I 12). Clearly, with 32P-labeled probes, fragments containing I could be found

In the sizes predicted by the cloning strategy. The recombinant virus, I

Formed by recombination of pSC11-P6, a negligible

In (about 500 bp), smaller HindIII fragment than the "pSC11-F41" recombinants (Fig. 1

In 12A and B). DNAs from uninfected cells showed no hybridization with the I

In 20 pSCl 1 nick-repair labeled probes or with the "Riboprobe" (Figs. 12A and B, lane I

I 9). DNA from the vSC8 virus hybridized only with the pSC11 "nick-repair" labeled I

In probe due to the common TK sequence and the lacZ gene (Fig. 12B, lane 8). FSME- I

As expected, the "Riboprobe" showed no hybridization. Both the "Riboprobe" and the I

However, in "nick-repair" labeled probes, "recombinant" identified HindIII J-I

In 25 fragments of different sizes (Figs. 12A and B, lanes 1-3 and 7). Vaccine DNA

In niaviruses that had arisen after recombination with the insertion plasmid pSC11 and I

In wild-type viruses, shown in FIG. 12A and B, lanes 4-6. With the "Riboprobe" (Fig. 12A) observe- I

In contrast, no hybridization was performed with the plasmid pSC11 (Fig. 12B). IN

I 30 15.11. Protein Analysis I

In Cells, infected with wild-type or recombinant viruses were infected with a m.o.i. of 1- I

In 10 pfu / cell. The cells were labeled with 35 S-methionine at various times I

I after the infection, harvested and analyzed one after the other on denaturing SDS-poly

35 acrylamide gels. Protein E variant molecules were determined by Western Blot Assay I

Claims (38)

The time course of synthesis of the FSME proteins in cells infected with vaccinia virus recombinants was determined by labeling the cells at different times after the infection for a short time (4 hours) with 35S-methionine. Since the p7.5 pro motor is active both in the early and late stages of the infection cycle, it was expected that the cells produced FSME proteins at an early and a late time point after infection. In comparison, the late arrow promoter regulates the synthesis of the β-gal protein having a molecular weight of 116 Kd. This protein therefore appeared relatively late after the infection. The described early or late promoter activities were evident at the time of harvesting of the cells, especially when larger amounts of virus were to be provided for purification and production of the 20 desired proteins. EXAMPLE 16 Vaccine Preparation 25 For the preparation of a vaccine, an antigen prepared according to the invention can be suspended, for example, in pure and effective form in a buffer solution and dissolved in an adjuvant in known manner to enhance immunogenic activity. 30 A DNA molecule, characterized in that it comprises DNA derived from the Western sub-type of FSME virus, which DNA encodes at least a portion of at least one protein selected from proteins C, prM, M or E of the western subtype of II FSME virus. IN DNA molecule according to claim 1, characterized in that it comprises the entire DNA sequence encoding the I I structural proteins of FSME virus, preferably as shown in FIG. 1. I DNA molecule according to claim 1 or 2, characterized in that it encodes one of the proteins selected from the proteins I, 10, C, prM, M or E of the western subtype of FSME virus. IN DNA molecule according to any one of claims 1-3, II characterized in that it encodes at least one region of an antigen-II determinant for one of the proteins selected from proteins C, prM, M or EII 15 of the western subtype of FSME virus, preferably the E protein. IN DNA molecule according to any one of claims 1 to 4, characterized in that it is obtained by reverse transcription with reverse I transcriptase from a purified RNA molecule of the western subtype of FSME-I I virus. IN DNA molecule according to any one of claims 1-4, characterized in that it is produced by chemical DNA synthesis. A DNA molecule according to any one of claims 1-6, characterized in that it hybridizes with a DNA molecule according to any one of claims 1-6 under stringent conditions. IN DNA molecule according to any one of claims 1-7, II characterized in that it comprises DNA sequences which differ from the DNA-II molecules of any one of claims 1-7 as a result of degeneration of II the genetic code and / or mutations and / or transpositions, but still encodes II for a protein having the essential antigenic properties of at least one of the II proteins selected from the proteins C, prM, M or E of the western sub-type of FSME virus. I DK 175515 B1 39 DNA molecule according to any one of claims 1-8, characterized in that it is combined with additional DNA sequences. A DNA molecule according to claim 9, characterized in that the additional DNA sequences allow replication and expression of the DNA molecule in a cell culture comprising sequences selected from promoters, enhancers, polyadenylation signals, and speciation signals. 10 RNA molecule, characterized in that it is derived from RNA from the western subtype of FSME virus and encodes at least a portion of at least one protein selected from the proteins C, prM, M or E of the western subtype of FSME virus. 15 An RNA molecule according to claim 11, characterized in that it comprises the entire RNA sequence encoding the structural proteins, preferably as shown in FIG. 2nd RNA molecule according to claim 11, characterized in that it encodes one of the proteins selected from proteins C, prM, M or E of the western subtype of FSME virus. RNA molecule according to any one of claims 11-13, 25, characterized in that it encodes at least one region from an antigenic determinant of one of the proteins selected from proteins C, prM, M or E of the western subtype of FSME virus, preferably the E protein. RNA molecule according to any one of claims 11-14, 30 characterized in that it is obtained by isolating and purifying RNA from the western subtype of FSME virus or by recombinant RNA / DNA techniques. 1 RNA molecule according to claim 15, characterized in that it is obtained by transcription of isolated and purified viral I RNA in DNA by reverse transcriptase and subsequent transcription of I in this DNA in RNA. IN RNA molecule according to any one of claims 11-15, characterized in that its complementary strand under stringent conditions hybridizes with an RNA molecule according to any one of claims 11-15. IN Vector, characterized in that it comprises insertions of DNA sequences according to any one of claims 1-10 or RNA sequences according to any of claims 11-17, the vector being selected from plasmids, viruses and cosmids. IN Vector according to claim 18, 15, characterized in that it is the plasmid pBR322, which contains a DNA sequence encoding a protein selected from proteins C, prM, M or E of the western subtype of FSME virus or parts. thereof. Vector according to claim 19, 20, characterized in that it is the plasmid A5 shown in FIG. 5 and which is deposited in DSM with the deposit number DSM 4382. Vector according to claim 19, characterized in that it is the plasmid P1-1 deposited in DSM with the deposition number DSM 4383. Vector according to claim 18 or 19, characterized in that it is an insertion plasmid which, by homologous recombination, transfers DNA sequences for the E protein or parts thereof into a HindIII J fragment from a vaccinia virus. Vector according to claim 22, characterized in that it is the plasmid pSC11-H1 of FIG. 7, which contains nucleotides 6503-8051 of the DNA sequence encoding the E protein. I 35 DK 175515 B1 41 Vector according to claim 22, characterized in that it is the plasmid pSC11-F41 of FIG. 7, which contains nucleotides 6503-7998 of the DNA sequence encoding the E protein. Vector according to claim 22, characterized in that it is the plasmid pSC11-P6 of FIG. 7, which contains nucleotides 6503-7524 of the DNA sequence encoding the E protein. Vaccine expression vector, characterized in that it contains, by homologous recombination, DNA sequences for the E protein, preferably from DNA sequences inserted into a plasmid according to any of claims 22-25, and capable of express protein E or portions thereof containing the amino acid sequences corresponding to said DNA sequences. 15 Carrier, characterized in that it comprises insertions of DNA sequences according to any one of claims 1-10 or RNA sequences according to any of claims 11-17, the carrier being a bacterium, preferably Salmonella typhi. 20 Cell culture, characterized in that it contains any of the sequences of any one of claims 1-17. Cell culture according to claim 28, characterized in that it allows expression of polypeptides encoded by the RNA or DNA sequences of any one of claims 1-17, the cell culture being preferably a mammalian cell culture. 1 35 Cell culture according to claim 28 or 29, characterized in that it is a Vero cell culture or a chicken embryo fibroblast cell culture containing a vaccinia expression vector according to claim 26, wherein the portion of the E protein corresponding to the inserted is expressed. DNA sequences. I DK 175515 B1 I I 42 I A peptide or polypeptide, characterized in that it comprises an amino acid sequence encoded by any of the nucleotide sequences of any one of claims 1- Peptide or polypeptide according to claim 31, characterized in that it is produced by expression of one of the sequences 11 according to any one of claims 1-17 in a suitable expression system. The peptide or polypeptide of claim 32, characterized in that it is produced in a cell culture according to claim 30. The peptide or polypeptide of claim 31, characterized in that it is chemically synthesized. I I 15 I Composition, characterized in that it comprises one or more of the peptides or poly-I peptides according to any one of claims 31-34. A live vaccine, characterized in that it comprises DNA sequences according to any one of claims 1-10 and / or RNA sequences according to any one of claims 11-11. 37. Live vaccine, characterized in that one or more DNA sequences according to any one of claims 1-10 are combined with vaccinia virus DNA. Live vaccine according to claim 37, characterized in that it contains a vaccine vector according to claim 26. 39. Vaccine, characterized in that it contains a composition according to claim 35. DK 175515 B1 43 Use of a vaccine according to any one of claims 36-39 for the preparation of specific immunoglobulins. Diagnostic agent, characterized in that it contains a composition according to claim 35. 42. DNA or RNA probe, characterized in that it hybridizes to DNA or RNA sequences according to any one of claims 1-17. 10