DE10218129A1 - Genetic vaccine against RNA virus infections - Google Patents

Abstract

The genetic vaccine is suitable for immunization against an infection with an RNA virus of genetic heterogeneity and comprises nucleotide sequences which code for proteins and / or protein fragments of this RNA virus. It is characterized in that the nucleotide sequences are different from one another, namely derived from gene segments of a plurality of different genotypes and / or subtypes of this RNA virus by means of transcription or translation and for proteins and / or protein fragments of this plurality of different genotypes and / or subtypes thereof Encode RNA virus.

Description

The invention relates to a genetic vaccine (a DNA vaccine) Immunization against infection with an RNA virus of genetic heterogeneity is suitable and which comprises the nucleotide sequences (nucleic acids) necessary for proteins and / or encode protein fragments of this RNA virus.

The invention also relates to a method for producing this vaccine.

The term "RNA virus with genetic heterogeneity" denotes RNA virus types that replicate with their own polymerase, which works with a relatively large error rate, as a result of which mutations are regularly incorporated into the virus genome. This also applies to retroviruses like HIV, which are among the viruses that replicate with single-stranded RNA and double-stranded DNA intermediate and an inaccurate RNA polymerase. The high mutation rate (e.g. for HCV by 1.4-1.9 × 10 ^-3 base changes per defined genome site per year) is characteristic of RNA viruses and leads to changes in the surface structure and the protein pattern of the virus envelope. These changes are the reason why the RNA viruses in question now have such a genetic diversity that today several (in the case of HCV six) different genotypes are classified in each of these virus types and a large number of subtypes are distinguished in each genotype. The biological consequence is that the RNA virus can no longer or only partially be recognized by the host's immune system in the course of the infection and the virus can escape the patient's immune response, namely neutralizing antibodies or cytotoxic T-lymphocytes. Secondly, the host is initially confronted with such a genetic diversity and thus surface variability of the RNA virus during virus inoculation that even a broad immune response is usually not sufficient to eliminate all genetically different genotypes and subtypes of this virus. By selecting mutants, the RNA virus can persist in spite of a clear immune response from the patient. In HCV, HIV, influenza and comparable infectious diseases or malignant tumors, this is regarded as one of the main mechanisms, if not the decisive mechanism, that leads to the chronification of the infectious disease or the permanent establishment of a tumor disease.

Infection with hepatitis C virus (HCV) is the most common cause of development post-transfusion or non-A-non-B hepatitis. The humoral and cellular The patient's immune response to HCV is polyclonal and multispecific. It is enough but for the reasons set out above, the virus usually does not last to eliminate. More than 70-80% of infected people stay after the exposure with HCV subsequently chronic virus carriers or develop chronic hepatitis. As the disease progresses, this chronic hepatitis can lead to Cirrhosis of the liver (in about 20-40% of virus carriers) or to a hepatocellular Lead to carcinoma. These complications go with significant morbidity and Mortality. Unfortunately, the therapeutic options have so far been limited. The Diverse quasi-species, subtypes and genotypes of HCV represent a big one Challenge to develop antiviral strategies because Immunization approaches but also virus inhibitors (e.g. NS3 protease inhibitors) are faced with a great genetic heterogeneity. So far, none protective hepatitis C vaccine are developed, which are a suitable agent for Curbing the spread of HCV.

In principle, the situation is very similar in the case of human Immunodeficiency Virus (HIV), Influenza Virus A, or Influenza Virus B infections.

The vaccine against RNA viruses comes in particular from the DNA vaccine and the Protein or peptide vaccine into consideration.

The principle of DNA vaccine or DNA immunization is based on the fact that a Plasmid that carries the genetic information for a desired target protein into the Patient cells is injected. The injection can be intracutaneous or intramuscular.

After the plasmid has been taken up into the cells, the desired endogenous product is endogenously Protein (s) expressed. These proteins or, if appropriate, from these by cleavage Peptides resulting from processing are located in the endoplasmic reticulum MHC molecules loaded and on the cell surface primarily in MHC class I Context presented. This leads to an MHC-restricted activation of CD8 + cytotoxic T lymphocytes (CTL), which play a major role in virus elimination play. At the same time, the expressed protein can be taken up by macrophages and induce an MHC class II response, which in turn stimulates T helper and B cells leads. Produce the stimulated T helper and B cells antigen-specific antibodies and thus the humoral immune response.

Investigation of the immune response after DNA immunization against the Structural proteins of HCV are known in the prior art. It has been shown that the HCV core protein, although highly conserved between the genotypes, not very much is immunogenic (see Geissler, M. et al., 1997 and Tokushige, K. et al., 1996). The published results of studies on immunogenicity against HCV Envelope proteins are controversial (cf. Salto, T. et al. 1997 and Nakano, I. et al. 1997), with these envelope proteins there is the problem of high genetic variability between genotypes and subtypes. The induction of a humoral and cellular Immune response to the non-structural proteins of HCV is also described (Encke, J. et. Al., 1998). However, none of these known test results provide a concrete reference point or even raw material for the development of an HCV Vaccine with at least satisfactory therapeutic properties.

In the development of protein or peptide vaccines as a prophylactic vaccine against RNA viruses there is the problem that these vaccines are essentially single MHC Class II Answer d. H. to induce antibody production, and that as a result immunization with recombinant protein of one or more RNA virus Genotypes and subtypes formed neutralizing antibody species according to the previous experience - especially with the example of HCV - is not sufficient to Variety of all RNA virus genotypes and subtypes are therapeutically effective fight. The DNA vaccine has the advantage that it primarily and first of all induces an immune response via the MHC class I pathway, namely one cytotoxic T cell response, plus an MHC class II response.

The object of the present invention is therefore to provide a vaccine or to develop a vaccine against RNA viruses which is suitable for the purpose of therapeutically effective immune response against a variety of different genotypes and to generate subtypes of the RNA virus in question and thus the further spread of RNA virus infectious diseases, in particular of hepatitis C virus (HCV), Human Immunodeficiency Virus (HIV), Influenza Virus A or Influenza Virus B Curb infectious diseases.

One solution to this problem is to provide a genetic vaccine of the type described in the introduction, which is characterized in that the Nucleotide sequences (or nucleic acids) are different from one another, namely from Genetic sections of a plurality (i.e. at least two) of different genotypes and / or subtypes of this RNA virus are derived by means of transcription or translation are and for proteins and / or protein fragments of this plurality different Encode genotypes and / or subtypes of this RNA virus.

In the present context, "derived" means that the nucleotide sequences completely or almost completely homologous or complementary to the concerned Gene segments are, with the deviations in individual nucleotide positions in the case of almost complete homology or complementarity to degeneration of the genetic code, or that the nucleotide sequences at least hybridize with these gene segments under stringent conditions.

The number of different RNA virus genotypes and / or subtypes is preferably about 10 to about 40.

In a preferred embodiment variant of the genetic according to the invention Vaccine, the different nucleic acids are different from gene segments known and characterized genotypes and / or subtypes of the RNA virus derived. This vaccine variant represents a so-called "defined Immunization Library "in which it is known which and how many different Genotypes / subtypes of the RNA virus in question in which amounts are present. An alternative to this is that variant of the Genetic vaccine according to the invention, in which the different nucleic acids of gene segments of different, in a pool of several, with regard to the concerned RNA virus positive patient sera of existing HCV genotypes and / or subtypes are derived. This vaccine variant represents a so-called "Undefined immunization library" because the vaccine contains an unknown Amount of genetic virus heterogeneity, i.e. H. here is not or at least not exactly known which and how many different genotypes / subtypes of the relevant RNA Virus in what quantities are present.

The different nucleotide sequences for the different proteins and / or Protein fragments of the various genotypes and / or subtypes of the subject RNA virus encoding are preferably inserted into a vector, either all or at least in groups in the same vector or individually or separately or isolated, d. H. each type for itself, one vector each. Both come as vectors Consider plasmids as well as adenovirein. In practice, the Plasmids pM-CG1b and pc DNA 3.1 proved to be well suited.

In addition to the vaccine, the invention also relates to a method for producing a genetic vaccine, which is characterized by the following process steps is: (a) isolation of virus RNA from a plurality of positive patient sera, (b) Amplification of the isolated virus RNA by means of PCR using Oligonucleotide primers which correspond to gene sections of the RNA virus genome, (c) ligation / insertion of the amplificates obtained into one or more vectors, preferably in one or more plasmids, cloning this recombinant Vectors / vectors or plasmids / plasmids, namely (d) transfection of competent Cells with this recombinant vector (s) or plasmid (s) and (e) selection and propagation of the transformed cells, (t) extraction of the Vector from the transformed cells using (preferably pyrogen-free) plasmid Mini, Mega or Giga preparation, (h) recording of the vectors in physiological Solution, preferably in physiological saline.

In a preferred embodiment of this method, plasmids are used as vectors used, preferably the or one of the plasmids pM-CG1b and / or pc DNA 3.1, and competent E. coli cells are used for the transfection according to step (d). Another advantageous variant of the method is characterized in that the in Virus RNA obtained in step (a) or the virus RNA obtained in step (b) Amplificates with regard to the genotype and / or subtype of the RNA virus in question is or are identified, and that the ligation according to step (e) is either in the the same vector or in different vectors. The hereby won Product is a so-called defined immunization library - in contrast to the so-called undefined immunization library, in which the different genotypes and / or subtypes of the RNA virus in question and therefore not identified are defined.

The genetic vaccine according to the invention or the genetic vaccine according to the invention For the first time, vaccine enables effective prophylactic immunization against a heterogeneous virus inoculum (i.e. against a variety of diverse RNA sequences or Sequence sections and proteins of a particular RNA virus), as is usually the case with a new infection with an RNA virus, for example an HCV, HIV or New influenza infection.

The vector or plasmid constructs of the DNA vaccine according to the invention can can be produced quickly, without special technical effort and inexpensively. In a large number of vector or plasmid constructs can be used in a relatively short time are constructed and cloned, which have the most diverse genetic Information from a large number of different genotypes or subtypes and / or Mutants of a certain RNA virus, in particular a hepatitis C virus (HCV), a human immunodeficiency virus (HIV), an influenza virus A or one Influenza B virus and consequently against at least all of these different Genotypes or subtypes and / or mutants of this particular RNA virus, in particular the hepatitis C virus (HCV) or the human immunodeficiency virus (HIV) or the influenza virus A or B a cellular and a humoral immune response can induce.

The DNA vaccine according to the invention also has the advantage that the vector DNA, in particular the plasmid DNA or the adenovirus stored at room temperature can be. This property also enables the use of such a vaccine in Third world countries.

The invention is described below using the example of a DNA vaccine against HCV Infections and DNA vaccines against HIV and influenza viruses explained in more detail. The examples include manufacturing and application processes with associated ones Characters.

Below the figures show:

Fig. 1 shows the location of the primers used for PCR within the HCV genome

Fig. 2 scheme for the preparation of the HCV constructs and cloning of the constructs in plasmids for the DNA vaccine

Figure 3 DNA vaccine constructs and vaccine groups

Fig. 4 immunization scheme for DNA vaccination

Fig. 5 ELISA (I) against recombinant HCV E1 protein

Fig. 6 ELISA (II) against a peptide mix of HCV E1

Fig. 7 T cell proliferation assay against HCV E1

Fig. 8 ELISPOT assay against HCV E1

example 1 Obtaining a "multigenetic" anti HCV DNA vaccine

The principle of the "multigenetic" anti-HCV DNA vaccine according to the invention exists therein, either (A) defined nucleotide sequences of a plurality of known genotypes and / or subtypes of HCV (a so-called "defined immunization library") in one integrate defined plasmid (insert, ligate) or (B) an unknown amount genetic virus heterogeneity (a so-called "undefined immunization library") to integrate into a defined plasmid (insert, ligate).

(A) To obtain the multigenetic anti-HCV DNA vaccine based A defined immunization library was operated as follows

HCV-positive patient sera isolated and characterized HCV-E1-RNA from various known and characterized HCV genotypes and subtypes according to Table 1 were used as starting material. This viral RNA was reverse transcribed and amplified by RT-PCR and subsequent nested PCR. The oligonucleotide primers (CS1, E2AS1, E1AS2, CS2 and E1AS2HIS) listed and characterized in Table 2 were used as primers, into each of which two restriction sites (EcoRV and HindII) and a start codon before the virus sequence (sense primer) and a stop Codon afterwards (antisense primer) were integrated. In order to reliably detect the various HCV genotypes and subtypes, partially degenerate primers were used. The location of the primers used for the PCR within the HCV genome is limited to the genome sections for the virus protein E1 by way of example and is shown in more detail in FIG. 1.

With this method, the HCV-E1 RNA amplificates (cDNA clones) A1, B1, C1, D1, F1, H77, I1, J1 and K2 won, the naming analogous to the naming of the Patient sera took place. In addition, an HCV-E1 RNA amplificate "MIX-Pos" obtained by an RNA mixture of all existing RNA samples of the RT-PCR has been subjected. This HCV-E1 RNA amplificate, designated as "MIX-Pos", provides is a defined gene library for HCV-E1, the viral heterogeneity in this Area. Approach H77 (genotype 1b) served as a positive control in the PCR. The various HCV-E1 RNA amplificates were in each case with the expert known and common methods in the plasmids pM-CG16 and pcDNA3.1. Zeo (-) ligated. Competent E. coli were then used with the recombinant plasmids transfected and the transformed cells selected and multiplied. As a control was a transfection with the "empty" plasmid, ie. the plasmid without inserted HCV-RNA amplificates (pM-CG16). Of the Transfection approaches "MIX-Pos" and "D" were 20 clones each, from all others Approaches picked 2 clones each and the (known to those skilled in the art) Subjugated to plasmid preparation. The success of the cloning was confirmed by a Restriction digestion and separation checked on an agarose gel.

The principle of this method for obtaining a multigenetic anti-HCV E1 DNA vaccine based on a defined immunization library is shown schematically in FIG. 2.

Positive bacterial clones from the mini preparation became a pyrogen-free plasmid Mega or Giga preparation used.

The plasmid-HCV-E1 constructs obtained were obtained by restriction digestion and Sequencing checked. The result of this control examination is in Table 3 shown.

In this Table 3 the sequences of the inserts of the plasmids pM-A1, pM-B1, pM-C1, pM-D1, pM-F2, pM-I1, pM-J1, pM-K2, pM-H77-1, pc-B-HIS and pc-J-HIS in direct Comparison and also lists the sequences of the PCR primers. The one in all plasmid sequence sections matched nucleotides are in the Table with a gray background. This comparison shows how much the different cloned HCV genotypes and subtypes at the nucleotide level differ.

All plasmid sequence sections shown have (in the 5'-3 'direction) Restriction interface EcoRV (GATATC) followed by a start codon (ATG) and the subsequent sequence of the sense primer CS2.

The plasmid HCV-E1 constructs derived from the vector pM-CG16 (pM-A1, pM- C1, pM-B1, pM-H77, pM-D1, pM-F2, pM-J1, pM-I1 and pM-K2) have in the plasmid sequence sections shown following the primer sequence a 639 Nucleotides (= 213 amino acids) long nucleic acid followed by a stop codon (TGA) and the subsequent restriction interface HindIII (AAGCTT). The of the vector pcDNA3.1. Zeo (-) derived plasmid-HCV-E1 constructs (pc-B-HIS and pc-J-HIS) have in the plasmid sequence sections shown in Following the primer sequence also a 639 nucleotide (= 213 amino acids) long nucleic acid followed by a stop codon (TGA). They also contain these two plasmid sequence sections between the end of the E1 sequence and the Stop codon a 6 × histidine sequence (CATCACCATCATCACCAT).

(B) To obtain the multigenetic anti-HCV DNA vaccine based an undefined immunization library was as follows method

In turn, isolated from HCV-positive patient sera served as the starting material HCV E1 RNA of various HCV genotypes and subtypes. In contrast to The procedure according to example (1A) was the HCV-positive in the present case Patient sera pooled from 20 different patients and from these pooled sera the HCV-E1 RNA isolated. The pooling was carried out in such a way that all clones from the 20th various patient sera had been obtained by sweeping over the Culture medium plate were introduced into a common bacterial culture approach. The The actual virus sequences are no longer with this procedure (1B) known, but it can be assumed that at least most, if not all, of the 20 pooled patient sera containing HCV-E1 RNA sequences from various HCV genotypes and HCV subtypes in the immunization library be mapped.

The pooled viral RNA obtained was nested using RT-PCR and subsequent PCR reverse transcribed and amplified. The one received and designated as "MIX-Lib" HCV-E1 RNA amplificate was known and familiar to the person skilled in the art Methods in the plasmids pM-CG16 and pcDNA3.1. Zeo (-) inserted (ligated) and cloned. The procedure was as described in Example (1A).

Competent E. coli were then transfected with the recombinant plasmids and selected. As a control, a transfection batch with the "empty" plasmid was used performed without inserted HCV-RNA amplificate (pM-CG16). Of the About 40 clones were picked from each of the transfection batches and the (to the expert known and common) subjected to plasmid mini-preparation. The success of Cloning was carried out by restriction digestion, separation on an agarose gel and sequencing checked.

The principle of this method for obtaining a multigenetic anti-HCV DNA vaccine based on an undefined immunization library is also shown schematically in FIG. 2.

Fig. 4 summarizes the different vaccination groups.

Example 2 Immunization of mice with the invention "Multigenetic" anti-HCV DNA vaccine

Analogously to the procedure described in Example 1, the HCV-E1-DNA plasmid constructs according to FIG. 3 and from them the two multigenetic anti-HCV-E1-DNA vaccines "MIX-pos" (defined immunization library) and "MIX-Lib "(undefined immunization library), as well as for comparison purposes the conventional" unigenetic "vaccines" pM-A-1 "," pM-B-1 "," pM-C-1 "," pM-D-1 "," pM- F-2 "," pM-I-1 "," pM-J-1 "," pM-K-2 "and" pM-H77-1 ". For this purpose, the plasmid DNA in question was taken up in physiological saline, namely in a concentration of 75 μg HCV-E1-DNA per 100 μl physiological saline. 13 groups of 10 Balb / c and C57 / Black mice each were pre-injected with 0.25% bupivacaine. Five days later, each mouse was injected intramuscularly into the thigh. This treatment was repeated twice every 14 days. The mice were sacrificed 12 days after the third immunization, the sera from each (vaccination) group were pooled and the humoral and cellular immune response by means of ELISA, T cell proliferation assay (CD4 + T cell response) and as a measure of the cytotoxic T cell response ( CD8 + T cells) measured with ELISPOT.

A recombinant, E. coli-expressed E1 protein, genotype I "HCV-E1 genotype-1" (from Biogenesis, UK) was used as the test antigen for the ELISA (I) and also for ex vivo stimulation in the other immunological assays , The results of the ELISA (I) are shown in FIG. 5. All mouse groups showed a clear humoral immune response, namely an induction of significant antibody levels. The antibody level after vaccination with the defined multigenetic anti-HCV E1 DNA vaccine (defined immunization library) "MIX-Pos" was significantly higher in almost all cases compared to the "unigenomic" vaccine. The antibody levels after vaccination with the undefined multigenetic anti-HCV E1 DNA vaccine (undefined immunization library) "MIX-Lib" were at least significantly above the background (after immunization with mock plasmid pM-CG-16).

A defined peptide mix of four defined peptides (prediction of the epitopes by computer program) with sequences which were taken from a genotype I served as test antigen for the ELISA (II). The results of the ELISA (II) are shown in Fig. 6. Only the mouse groups that had been vaccinated or immunized with the "unigenetic" vaccine against HCV genotypes IV and V, namely the groups "pM-I-1", "pM-J-1" and "pM-K- 2 "(cf. Tab. 1), and the mouse group which had been vaccinated or immunized with the defined multigenetic anti-HCV-E1 DNA vaccine (defined immunization library)" MIX-Lib "showed a clear humoral immune response, namely an induction of significant antibody levels.

In Fig. 7 shows the results of T-cell proliferation assays against HCV are shown E1, namely, the proliferation index and stimulation index of CD4 + T-cells after three days of in vitro stimulation with the above-described recombinant protein "HCV E1 genotype 1 "(Biogenesis, UK). A proliferation index or stimulation index greater than 2 was only detectable in the mouse group that had been vaccinated or immunized with the "unigenetic" vaccine against the HCV genotypes V ("pM-K-2"). However, significantly high T cell proliferation levels were also found in those mouse groups which had been vaccinated or immunized with the defined multigenetic anti-HCV E1 DNA vaccines (defined immunization libraries) "MIX-Pos" and "MIX-Lib" ,

In FIG. 8 shows the results of ELISPOT assays against the genotype I HCV E1 recombinant protein are (Fa. Biogenesis, UK), respectively. The ELISPOT shows the number of IFN-gamma producing T cells at the individual cell level and is therefore considered a relative measure of the cytotoxic T cell response. After immunization with the multigenetic and unigenetic vaccines described, a significantly high CD8-T cell activity was found in those mouse groups which were treated with the multigenetic anti-HCV-E1 DNA vaccines (immunization libraries) "MIX-Pos" and "MIX-Lib" had been vaccinated or immunized, and also in the mouse group which had been vaccinated or immunized with the "unigenetic" vaccine against the HCV genotypes Ib ("pM-H77-1").

Overall, the results of immunological experiments show genetic Immunization against E1 clearly shows that the multigenetic according to the invention Vaccines of different genotypes of different epitopes of different or the same Recognize genotypes, induce overarching immune responses and thus for one successful HCV control are suitable.

Example 3 Obtaining a "multigenetic" vaccine according to the invention against HIV and a "multigenetic" according to the invention Influenza virus vaccine

Envelope proteins of HIV or influenza virus of different genotypes and Subtypes are analogous to the DNA vaccine for HCV (see Example 1) in a plasmid or cloned an adenovirus. For influenza, hemagglutinin and Neuraminidase genes of various subtypes used (isolated from patient serum or birds or mammals in Southeast Asia). In HIV, gp120 is considered Membrane protein with high variability used in addition to other membrane proteins (Cloning from patient sera analogous to HCV). Via degenerate primers at the ends these proteins are converted into a plasmid or viral gene segment using RT-PCR Vector introduced and subsequently checked in vitro by Western blot analysis. to Expression or genotype-independent expression analysis with immunoblot becomes a myc tag introduced into the plasmid.

Example 4 Immunization of mice with the invention "multigenetic" anti-HIV DNA vaccine or with the "multigenetic" anti-influenza virus DNA according to the invention vaccine

Analogous to the procedure described in Example 2, BALB / c and C57 / BL6 Mice multiple times intramuscularly and / or subcutaneously with that obtained according to Example 3 DNA vaccine immunized against HIV or against influenza virus. The measurement of humoral immune response is via an ELISA. In doing so, proteins different genotypes and subtypes or published in the literature Peptide sequences of different subtypes of HIV or influenza virus are used. Cross-subtype humoral immune responses with cross-subtype neutralizing antibodies demonstrate the broad genetic heterogeneity associated with a such vaccination according to the invention is covered. The same applies to the cellular Immune response. Here, the CD4 T cell response in the thymidine proliferation assay or in ELISPOT and the CD8 cytotoxic T cell response via with the adenoviruses infected target cells measured. Both play for virus elimination, above all also in patients who are already infected (use as a therapeutic vaccine), one decisive role. In the mouse model, the described immunizations are used genotype / subtype generated broad immune response that the virus Immune evasion via genetic mutation (which occurs after conventional vaccinations) makes impossible. There can be no or only very small amounts of so-called escape mutants arise.

Example 5 Immunization of monkeys with an inventive "multigenetic" DNA vaccine

In a virus challenge model in monkeys, the prophylactic effectiveness of the "multigenetic" DNA vaccine obtained according to Example 1 or Example 3 against HCV or HIV or influenza virus is demonstrated by successfully eliminating the challenge isolate from various genotypes and subtypes. References 1. Saito, T. et al .: Plasmid DNA-based immunization for hepatitis C virus structural proteins: immune responses in mice. Gastroenterology 112, 1321-30 (1997).
2. Geissler, M. et al .: Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokineexpressing plasmids. J Immunol 158, 1231-7 (1997).
3. Tokushige, K. et al .: Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology 24, 14-20 (1996).
4. Nakano, I. et al .: Immunization with plasmid DNA encoding hepatitis C virus envelope E2 antigenic domains induces antibodies whose immune reactivity is linked to the injection mode. J Virol 71, 7101-9 (1997).
5. Encke, J. et al .: Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in a murine model. J Immunol 161, 4917-23 (1998). Table 1 Genotypes of the (patient) sera used for cloning

Table 2 Oligonucleotide primers used for PCR

Table 3 Sequence comparison of the inserted HCV constructs in the plasmids for the DNA vaccine

Claims (17)

1. Genetic vaccine which is suitable for immunization against infections with an RNA virus and comprises nucleotide sequences which code for proteins and / or protein fragments of this RNA virus, characterized in that the nucleotide sequences are different from one another, namely from gene segments of a plurality of different ones Genotypes and / or subtypes of this RNA virus are derived by means of transcription or translation and code for proteins and / or protein fragments of this plurality of different genotypes and / or subtypes of this RNA virus. 2. Genetic vaccine according to claim 1, characterized in that that the different nucleic acids from different known gene segments and characterized genotypes and / or subtypes of the RNA virus are derived. 3. Genetic vaccine according to claim 1, characterized in that the different nucleic acids of different gene segments, in one from several virus positive patient sera of existing genotypes and / or Subtypes of the RNA virus are derived. 4. Genetic vaccine according to one of claims 1 to 3, characterized in that the different nucleotide sequences that are for the different proteins and / or protein fragments of the different genotypes and / or subtypes of the Encode RNA virus, are inserted in a vector plasmid. 5. Genetic vaccine according to claim 4, characterized in that the different nucleotide sequences that are for the different proteins and / or protein fragments of the different genotypes and / or subtypes of the Encode RNA virus inserted in the same vector. 6. Genetic vaccine according to claim 4, characterized in that the different nucleotide sequences that are for the different proteins and / or protein fragments of the different genotypes and / or subtypes of the Encode RNA virus, inserted individually in each vector. 7. Genetic vaccine according to one of claims 1 to 6, characterized in that the vector is a defined plasmid, preferably the plasmid pM-CG1b or pc DNA 3.1 Zeo (-). 8. Genetic vaccine according to one of claims 1 to 6, characterized in that the vector is an adenovirus. 9. A method for producing a genetic vaccine, characterized by the following steps: a) isolation of the virus RNA from a plurality of positive patient sera, b) amplification of the isolated virus RNA by means of PCR using oligonucleotide primers which correspond to sections of the RNA virus genome, c) ligation / insertion of the amplificates obtained into one or more vectors, d) transfection of competent cells with the recombinant vector (s) from (c), e) selection and propagation of the transformed cells from (d); f) obtaining the vector (s) from the transformed cells by means of plasmid mini, mega or giga preparation, g) Recording the vectors in physiological solution. 10. The method according to claim 8, characterized in that the virus RNA obtained in step (a) or that obtained in step (b) Virus RNA amplificates with regard to the genotype and / or subtype of the RNA Virus are identified and that the ligation according to step (e) in the same Vector is done. 11. The method according to claim 8, characterized in that the virus RNA obtained in step (a) or that obtained in step (b) Virus RNA amplificates with regard to the genotype and / or subtype of the RNA Virus are identified and that the ligation according to step (e) into different Vectors. 12. Genetic vaccine according to one of claims 1 to 8, characterized in that the RNA virus is an HCV virus. 13. Genetic vaccine according to one of claims 1 to 8, characterized in that the RNA virus is an HIV virus. 14. Genetic vaccine according to one of claims 1 to 8, characterized in that that the RNA virus is an influenza virus. 15. The method according to any one of claims 9 to 11, characterized in that that the RNA virus is an HCV virus. 16. The method according to any one of claims 9 to 11, characterized in that that the RNA virus is a HiV virus. 17. The method according to any one of claims 9 to 11, characterized in that the RNA virus is an influenza virus.