HK1163744A - Flavivirus ns1 subunit vaccine - Google Patents

Description

Flavivirus NS1 subunit vaccine

The application is a divisional application of the invention with the application date of 2002, 11/20/h, Chinese application No. 02824307.2 and the invention name of 'flavivirus NS1 subunit vaccine'.

The present invention relates to the NS1 protein of flavivirus, in particular dengue virus, or a part thereof, which can be used for vaccination against said flavivirus and against one or more other flaviviruses. The invention further relates to NS1 protein or a part thereof from one dengue virus serotype, in particular serotype 2, that can be used against all serotypes of dengue virus by vaccination. The invention further relates to DNA containing an expression module encoding flavivirus NS1 or a part thereof, vectors containing said DNA and vaccines containing or expressing flavivirus NS 1.

Background

The causative agent of dengue fever is the dengue virus, which belongs to the flavivirus genus of the Flaviviridae family (Burke and Monath, 2001). A very important subgroup of flaviviruses is the population known as mosquito-borne flaviviruses, i.e. the group of flaviviruses transmitted by mosquitoes. This group includes, in addition to the above dengue viruses, other important viruses such as West nile virus (West nile virus), Japanese encephalitis virus (Japanese encephalitis virus) and yellow fever virus (Fields Virology, ed.by Fields B.N., Lippincott-Raven Publishers, 3rd edition 1996, ISBN: 0-7-. Typical diseases transmitted by these viruses are West Nile fever and West Nile encephalitis caused by West Nile virus, encephalitis caused by Japanese encephalitis virus, yellow fever caused by yellow fever virus and dengue virus, dengue hemorrhagic fever caused by dengue virus (DHF; see below) and Dengue Shock Syndrome (DSS).

Flaviviruses are enveloped single-stranded positive-stranded RNA viruses consisting of three structural proteins: a capsid protein (C) that binds to the viral genome forming a nucleocapsid surrounded by a lipid bilayer anchored with M (membrane) and E (envelope) proteins. The genome is approximately 11kb and comprises a single open reading frame encoding a polyprotein precursor of about 3400 amino acid residues. Each viral protein is produced from this precursor by the action of cellular and viral proteases. Three structural proteins (C, M and E) are derived from the N-terminal part of the polyprotein, which are followed by seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5(Lindenbach and Rice, 2001).

The glycoprotein NS1 present in all flaviviruses tends to be essential for virus viability. Dengue virus NS1 is secreted from mammalian infected cells as a soluble hexamer (Flaland et al, 1999). This non-covalently bound hexameric complex is formed from 3 dimeric subunits, having a molecular weight of 310 kDa. Dimerization is a prerequisite for export of the NS1 protein to the plasma membrane, where it serves as the only viral resident protein on the surface of infected cells.

In mammalian cells, the transported fraction of NS1 was released into the extracellular environment, but not in dengue-competent insect cell lines. Extracellular NS1 is secreted as a soluble protein, either in higher hexameric form or bound to microparticles and not to virions. Furthermore, NS1 has been found to circulate in the serum of patients infected with dengue virus, suggesting that NS1 secretion may be an important event in the course of flavivirus infection in human hosts. During flavivirus infection, the NS1 protein elicits a strong antibody response that aids in the clearance of the infecting virus from the host, presumably through complement-mediated pathways (Schlesinger, j.j. et al, 1987) and antibody-dependent cellular cytotoxicity (ADCC) (Schlesinger, j.j. et al, 1993).

There are four serotypes of dengue virus: dengue virus serotype 1(Den-1) to dengue virus serotype 4(Den-4), the most important member of the flavivirus genus involved in infecting humans, causes diseases ranging from cold-like symptoms to severe or fatal diseases, dengue hemorrhagic fever and shock syndrome. Dengue outbreaks have been a major public health problem in densely populated areas of the tropics and subtropics where mosquitoes are abundant.

Much concern has been placed in many areas of the world about the spread of dengue infection and other diseases caused by mosquito-borne flaviviruses, and as a result, dengue vaccines have been vigorously developed that can prevent Dengue (DF) and Dengue Hemorrhagic Fever (DHF), and have been available that are useful for protecting vaccinated individuals against infection caused by some or all of the mosquito-borne flaviviruses.

Most cases of DF are symptomatic after a first infection with any one of the four serotypes, with the majority of cases of DHF occurring in subjects infected a second time with a serotype different from the serotype of the dengue virus that was first infected. These observations lead to a hypothesis: continued infection of individuals who have had antibodies against one dengue serotype with another virus serotype at appropriate intervals may cause a significant number of cases of DHF. Antibody-dependent enhancement (ADE) of dengue virus as well as other enveloped viruses has been demonstrated in vitro and is considered to be an important mechanism in the etiology of DHF.

DHF has been found to occur generally in geographical areas where multiple (three or four) virus serotypes are co-circulating. In regions with endemic DHF, such as southeast asian countries, age-specific incidence is higher in children and the number of DHF cases is reduced in the older age group. This roughly corresponds to an increasing rate of dengue seropositivity, indicating that natural infection may have elicited protective immunity. This phenomenon is unlike other viral infections observed, such as hepatitis a virus. Clinical observations from a control-free case description show that patients may experience DHF twice (nimmnitya et al, 1990), but this is rare and it is very difficult to correctly identify the serotype responsible for the second and subsequent infections. To date, although all four dengue virus serotypes are circulating in the same region, there has been no report of late stage infection in the same individual. This suggests that in nature, infection with two or three dengue virus serotypes in the same individual may form cross-reactive antibodies and even cross-reactive cytotoxic lymphocyte responses. This may modulate or protect the body against infection by dengue virus serotypes that persist in nature.

There is currently no approved dengue vaccine. Prevention of dengue virus infection is now dependent on the control of the major mosquito vector Aedes aegypti (Aedes aegypti). Resistance to insecticides, lack of technical and economic support to enable local health authorities to maintain effective mosquito control programs, and the continued regional spread of mosquito and dengue viruses as vectors make resistance to dengue infection virtually impossible with current mosquito control programs. Therefore, the development of safe and effective vaccines against all four serotypes of dengue virus has been designated by the World Health Organization (WHO) as the most cost-effective method to focus on the prevention of dengue virus infection. The world health organization recommends that the ideal vaccine against dengue fever and DHF should be one that prevents infection by all serotypes, so that continuous infection cannot occur.

To this end, WO98/13500 proposes the use of one recombinant modified vaccinia virus ankara (MVA) expressing antigens from all serotypes of dengue virus, and the use of four recombinant MVAs, each of which expresses at least one antigen of one dengue virus serotype. Both of these strategies provide a very promising strategy for vaccination against all serotypes of dengue virus. However, it would be desirable to provide a single subunit vaccine which, upon administration, elicits an immune response against more than one flavivirus or more than one dengue virus serotype, preferably against all dengue virus serotypes. WO98/13500 also discloses a recombinant MVA encoding dengue virus NS 1. WO98/13500 does not disclose that an antigen derived from one dengue virus serotype is capable of eliciting an immune response against not only the dengue virus serotype producing the antigen, but also against antigens derived from other dengue virus serotypes.

WO99/15692 discloses a recombinant MVA comprising and capable of expressing one or more DNA sequences encoding dengue virus antigens that do not cause immune enhancement or antibody-dependent enhancement. WO99/15692 does not disclose that an antigen derived from one dengue virus serotype elicits an immune response against not only the dengue virus serotype producing the antigen, but also antigens derived from other dengue virus serotypes.

Object of the Invention

It is an object of the present invention to provide a vaccine derived from a flavivirus or flavivirus serotype that is stable, easy to produce, and capable of eliciting an immune response that protects the vaccinated individual not only against the flavivirus or flavivirus serotype from which the vaccine was derived, but also against other flaviviruses or flavivirus serotypes. It is a particular object of the present invention to provide a vaccine derived from a mosquito-borne flavivirus that protects the vaccinated individual not only against the mosquito-borne flavivirus or flavivirus serotype from which the vaccine is derived, but also against other mosquito-borne flaviviruses or flavivirus serotypes. It is another object of the present invention to provide a vaccine derived from one serotype of dengue virus that protects vaccinated individuals against infection by at least two, preferably all, serotypes of dengue virus.

Detailed Description

These problems are solved by the flavivirus NS1 protein or a part thereof, respectively, and a DNA sequence comprising an expression module encoding the flavivirus NS1 protein or a part thereof. In particular, by utilizing the NS1 protein or a portion thereof of a mosquito-borne flavivirus, particularly a dengue virus, preferably dengue virus serotype 2, and the corresponding DNA sequences, a vaccine derived from one mosquito-borne flavivirus is provided that protects an individual against infection by the mosquito-borne flavivirus from which the vaccine is derived, and against infection by at least one other mosquito-borne flavivirus. More particularly, by utilizing the NS1 protein or a portion thereof and the corresponding DNA sequence of a dengue virus, particularly dengue virus serotype 2, a vaccine derived from one dengue virus serotype is provided that protects an individual against infection by at least two, preferably at least three, more preferably all serotypes of the dengue virus, and preferably also against infection by other flaviviruses, particularly mosquito-borne flaviviruses such as Japanese encephalitis virus, yellow fever virus and West Nile virus.

As shown in more detail in the experimental section, NS1 protein, derived from one serotype of dengue virus and re-expressed after vaccination, is capable of eliciting an antibody response that cross-reacts with the NS1 protein of dengue virus serotypes 1, 2, 3 and 4 and the NS1 protein from other members of the flavivirus genus, such as Japanese encephalitis virus, yellow fever virus and West Nile virus. Thus, the NS1 protein from one dengue virus serotype is a universal DHF subunit vaccine against at least two, more preferably three, even more preferably all four dengue virus serotypes simultaneously, but also against one or more other viruses of the flavivirus genus. Since the E protein is not included in this subunit vaccine strategy, there will be no risk of antibody-dependent enhancement (ADE) upon subsequent exposure to any one of the dengue serotypes, and will not cause vaccine-associated DHF upon natural outbreak of dengue infection.

The NS1 protein may be expressed from a nucleic acid, preferably DNA comprising an expression module encoding at least one flavivirus NS1 protein or portion thereof. The term "at least" is to be construed herein as meaning that the expression module may additionally encode other proteins/peptides, either alone or fused to the NS1 protein or a portion thereof, as defined in more detail below. The term "DNA" in the present invention refers to any form of DNA, such as single-stranded DNA, double-stranded DNA, linear or circular DNA, or DNA in the form of a plasmid or viral genome. Since flaviviruses are RNA viruses, the DNA encoding the flavivirus NS1 protein is non-natural DNA, such as cDNA or synthetic DNA.

The term "expression cassette encoding a flavivirus NS1 protein or portion thereof" is to be construed as a sequence encoding the flavivirus NS1 protein or portion thereof preceded by elements controlling transcription, particularly transcription initiation. Examples of such transcriptional regulatory elements are prokaryotic promoters and eukaryotic promoters/enhancers. Preferred eukaryotic promoters/enhancers are the human cytomegalovirus immediate early promoter/enhancer and poxvirus promoters, such as the 7.5 promoter and poxvirus minimal promoter, see examples section. The sequence of the poxvirus minimal promoter is shown in figure 2 and SEQ: ID No. 9. If desired, the expression module may further comprise elements controlling the termination of transcription, such as prokaryotic termination elements or eukaryotic PolyA signal sequences.

The expression module may express the flavivirus NS1 protein or a portion thereof alone or NS1 protein or a portion thereof along with one or more other flavivirus proteins/peptides, wherein NS1 protein or a portion thereof and the other proteins/peptides are made as separate proteins/peptides or as a fusion protein. The term "peptide" in the present invention refers to a contiguous amino acid sequence of at least 10 amino acids, more preferably at least 20 amino acids, most preferably at least 25 amino acids, if not otherwise defined in the specification.

The other flavivirus protein is not the entire E protein, as this protein appears not to be involved in the progression of DHF. Thus, if the other flavivirus peptide is derived from the E protein, it will comprise less than 40 amino acids, preferably less than 35 amino acids. If the amino acid sequence derived from the E protein is expressed together with the NS1 protein or a part thereof, it should be confirmed that this amino acid chain does not contain an epitope involved in the occurrence of ADE and DHF.

If the expression module expresses other flavivirus proteins/peptides in the form of separate proteins/peptides in addition to the NS1 protein or part thereof, the expression module may comprise an Internal Ribosome Entry Site (IRES) between the sequence encoding the NS1 protein or part thereof and the sequence encoding said other flavivirus protein. IRES elements are well known to those skilled in the art. Examples of IRES elements are picornavirus IRES elements or the 5' non-coding region of hepatitis C virus.

In addition, the nucleic acid sequence encoding the NS1 protein or portion thereof may be fused to DNA sequences encoding other flavivirus proteins/peptides to produce a fusion protein of the NS1 protein or portion thereof and the other flavivirus proteins/peptides. If the NS1 protein or portion thereof is to be produced as a fusion protein/peptide with other flavivirus proteins/peptides, its corresponding coding sequence is fused in-frame.

In a preferred embodiment, the DNA sequence encoding the NS1 protein or a portion thereof is preceded by a sequence encoding an E protein glycosylation signal. According to this embodiment, a fusion protein comprising the glycosylation signal sequence of the E protein fused to the NS1 protein or a portion thereof was prepared. As indicated above, the E protein-derived amino acid chain should be as short as possible and it should not include epitopes involved in the development of ADE and DHF. The glycosylation signal sequence of the E protein meets these requirements.

In another preferred embodiment, the expression module of the invention comprises a sequence encoding the NS1 protein or a portion thereof as the only flavivirus sequence. Thus, in this preferred embodiment, the expression module according to the invention does not express any other peptides/proteins of other parts of the flavivirus genome, in particular does not express NS2A or E proteins.

In another preferred embodiment, the DNA of the invention expresses a fusion protein of the NS1 protein or a portion thereof with a protein/peptide not derived from a flavivirus comprising a non-flavivirus signal sequence or a sequence such as a tag that can be used to detect or purify the expressed fusion protein.

In order to understand the general structure of flavivirus sequences in the expression cassette used in a preferred embodiment of the present invention, it is advantageous to briefly summarize the structure of the flavivirus genome: during natural flavivirus infection, the virus produces a polyprotein that is cleaved by host cell proteases and then by virus-encoded proteases into the following proteins: c, PrM and M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (protein order on the polyprotein precursor). Thus, the DNA sequence, particularly the cDNA sequence, encoding the NS1 protein or a portion thereof, must require the addition of an "ATG" initiation codon. In a preferred embodiment, the initiating ATG is followed by a sequence encoding a glycosylation signal to glycosylate the newly synthesized NS1 protein in the endoplasmic reticulum. Such signal sequences are well known to those skilled in the art. Finally, the protein coding module requires a stop codon, which may be TAG added to the 3' end of the cDNA sequence encoding the protein. The "ATG + signal sequence" element used in the examples of the invention was derived from the sequence encoding the hydrophobic C-terminus of the E protein (the last 28 amino acids, starting at amino acid m (ATG)) in the dengue virus New guineaa strain ("NGC strain", GeneBank accession No. AF 038403). Typical expression modules according to the invention are shown in fig. 2, SEQ: ID NO 9 and SEQ: ID NO 10.

Thus, in general terms, this embodiment relates to the use of DNA containing expression cassettes encoding the flavivirus NS1 protein or portions thereof, wherein the coding sequence is preceded by an initiation codon ("ATG") and a sequence encoding a glycosylation signal sequence, preferably a sequence derived from the E protein as defined above, and the coding sequence ends at a translation stop codon (FIGS. 1A, 1C and 2, SEQ: ID 5-10).

The DNA sequence of the present invention encodes the flavivirus NS1 protein or a portion thereof. The term "flavivirus" refers to any flavivirus. More preferably, the term "flavivirus" refers to mosquito-borne flaviviruses such as West Nile virus, Japanese encephalitis virus, yellow fever virus and dengue virus. The NS1 protein or portion thereof encoded by the DNA of the invention that is derived from a mosquito-borne virus should protect vaccinated individuals not only against infection by the virus or serotype from which the vaccine was derived, but also against infection by other mosquito-borne viruses or other serotypes of the virus from which the vaccine was derived. The NS1 protein is preferably any dengue virus serotype. More preferably the NS1 protein coding sequence is derived from dengue virus serotype 2, such as the dengue virus New Guinea strain ("NGC strain", GeneBank accession No. AF 038403). The terms "subtype" and "serotype" are used interchangeably in the specification.

The term "part thereof" in the term "NS 1 protein or a portion thereof" herein refers to a stretch of amino acid chains of the NS1 protein that is long enough to induce a specific immune response against the NS1 protein from which the "part thereof" is derived. If the flavivirus is dengue virus, the amino acid chain should be capable of eliciting an immune response against the NS1 protein of all serotypes of dengue virus in vaccinated animals (including humans). In the examples section, it is shown how a person skilled in the art can determine whether the NS1 protein or a part thereof elicits a specific immune response against all serotypes of dengue virus. According to a preferred embodiment, the flavivirus DNA sequence encodes the entire NS1 protein. Thus, the term "NS 1 protein or a portion thereof" refers to the entire sequence of the native NS1 protein, as well as shorter epitope chains that still elicit an immune response.

Furthermore, the term "NS 1 protein" also refers to derivatives of the native NS1 protein. The derivative may be a protein obtained by substituting, deleting and/or inserting one or more amino acids in the native NS1 protein. By way of example, the derivatives are proteins having an amino acid sequence homology of at least 50%, preferably at least 75%, more preferably at least 90%. Thus, the term "part thereof" also refers to the part of the NS1 protein derivative.

In summary, a most preferred embodiment of the invention is the use of a DNA comprising an expression cassette encoding a mosquito-borne flavivirus NS1 or part thereof, wherein the flavivirus is preferably dengue virus, especially dengue virus serotype 2, and wherein the expression of the NS1 protein or part thereof is controlled by transcriptional regulatory elements. More preferably, the DNA of the invention encodes the NS1 protein or a part thereof in the form of a fusion protein with a glycosylation signal sequence.

The invention further relates to a vector comprising a DNA as described above, and to the use of said vector for inducing an immune response according to the invention. The term "vector" refers to any vector known to those skilled in the art. The vector may be a plasmid vector such as pBR322, or a pUC series vector. More preferably, the vector is a viral vector. In the context of the present invention, the term "viral vector" or "viral vector" refers to an infectious virus containing a viral genome. In this case, the DNA of the invention is cloned into the viral genome of the corresponding viral vector. The recombinant viral genome is then packaged, and the resulting recombinant vector can be used to infect cells and cell lines, particularly living animals (including humans). Typical viral vectors that may be used according to the invention are adenoviral vectors, retroviral vectors, or vectors based on adeno-associated virus 2(AAV 2). Most preferred are poxvirus vectors. The poxvirus is preferably a canarypox (canarypox) virus, an avipox virus or a vaccinia virus. More preferably modified vaccinia virus Ankara (MVA) (Sutter, G.et al, 1994, Vaccine 12: 1032-40). A typical MVA strain is MVA-575 which has been deposited at the European Collection of animal cells (ECACC) located in Wiltshire, UK on 12/7.2000 under the accession number ECACC V00120707. Most preferred is MVA-BN or a derivative thereof as described in PCT application WO02/42480 (PCT/EP 01/13628). The contents of the above applications are included in the present application as references. MVA-BN has been deposited at 30/8/2000 at the european collection of animal cells located in wiltshire, uk under the accession number ECACC V00083008. Since the MVA-BN viral vector is a highly attenuated virus, additional technical problems are solved by using MVA-BN or derivatives thereof, thereby providing a particularly safe viral vaccine against flaviviruses. In particular, MVA-BN has been demonstrated to be less virulent than MVA strains known in the prior art. MVA-BN is derived from modified vaccinia ankara and has the property of losing its ability to replicate in human cell lines. MVA-BN is much safer than any other known vaccinia strain due to its inability to replicate in humans. In a preferred embodiment, the present invention relates to a viral vector comprising DNA of MVA-BN and MVA-BN derivatives as described above. Characterization of MVA-BN, biological assays to assess whether a MVA strain is MVA-BN or a derivative thereof and methods of obtaining MVA-BN or a derivative thereof are disclosed in WO 02/42480.

As used herein, a "derivative" of the virus with the deposit number ECACC V00083008, i.e. a derivative of MVA-BN, is as defined in WO 02/42480. The properties of the MVA-BN derivatives are briefly described below. For more detailed information on the definition of MVA-BN derivatives, in particular on the biological assays used to determine whether MVA viruses are the mentioned MVA-BN derivatives see WO 02/42480. Thus, the term refers to vaccinia viruses that exhibit at least one of the following characteristics of the MVA-BN deposit, but differ in one or more places in their genome. Preferred derivatives have at least two, more preferably at least three, most preferably all of the following four MVA-BN characteristics:

reproductive replication in Chicken Embryo Fibroblasts (CEF) and in baby hamster kidney Cell line BHK (ECACC 85011433), but not in human Cell line HaCat (Boukamp et al 1988, J Cell biol.106 (3): 761-71),

the inability to replicate in vivo,

-inducing a higher immunogenicity in a lethal challenge model compared to the known strain MVA 575(ECACC V00120707), and/or

-inducing at least substantially the same level of immunity in a vaccinia virus prime/vaccinia virus boost regimen as compared to a DNA prime/vaccinia virus boost regimen.

In particular, the derivative of MVA-BN has substantially the same replication characteristics as MVA-BN. Viruses with the same "replication characteristics" as the deposited viruses were those that replicated with similar amplification ratios in CEF cells and cell lines BHK, HeLa, HaCat and 143B and were determined to have similar replication in the transgenic mouse model AGR 129.

The term "incapable of reproductive replication" as used in the present application is as defined in WO 02/42480. Thus, a virus that is "incapable of reproductive replication" refers to a virus that amplifies less than 1 in the human Cell line HaCat (Boukamp et al 1988, J Cell biol.106 (3): 761-71). Preferably, the viruses used as vectors of the invention have an amplification ratio in the human cell line HaCat of 0.8 or less. "amplification ratio" of a virus refers to the ratio of the amount of virus produced by an infected cell (output) to the amount of virus originally used to infect the cell (input) ("amplification ratio"). A ratio of "1" of the output amount to the input amount indicates an amplified state in which the amount of virus produced by the infected cells is equal to the amount of virus originally used for the infected cells. .

In the context of the definition of MVA-BN and derivatives thereof, the term "replication in vivo" as used herein is the same as defined in WO 02/42480.Thus, the term is used in the context of WO02/42480 to refer to those viruses which are incapable of replication in human and mouse models. The mice used in WO02/42480 do not give rise to mature B-cells and T-cells (AGR 129 mice). Specifically, MVA-BN and its derivatives were injected intraperitoneally 10 into AGR129 mice⁷Mice are not killed for at least 45 days, preferably for at least 60 days, most preferably for at least 90 days after infection with pfu viruses. Preferably, another feature of the "replication incompetent in vivo" virus is that AGR129 mice are injected intraperitoneally with 10⁷pfu virus cannot be harvested from the mouse organ or tissue for at least 45 days after infection, preferably at least 60 days, most preferably at least 90 days.

MVA-BN and derivatives thereof are preferably tested in the lethal challenge mouse model described in WO02/42480, with an immunogenicity higher than that of the known diseased strain MVA 575. In such a model, unvaccinated mice were killed after infection with replication competent vaccinia strains such as Western Reserve strain L929TK + or IHD-J. In the context of describing a lethal challenge model, infection with replication-competent vaccinia virus is a process referred to as "challenge". 4 days after challenge, mice were generally killed and virus titers in the ovaries were determined by standard plaque assay using VERO cells. Virus titers were determined in unvaccinated mice, as well as in mice vaccinated with vaccinia virus of the invention. More specifically, MVA-BN and derivatives thereof were inoculated 10 in the above assay²TCID₅₀The mouse ovarian virus titer is reduced by at least 70%, preferably at least 80%, more preferably at least 90% for the virus/ml compared to no inoculation of the virus.

MVA-BN or derivatives thereof preferably have the following properties: at least substantially the same level of immunity is induced in the vaccinia virus prime/vaccinia virus boost regimen as compared to the DNA prime/vaccinia virus boost regimen. When the CTL response is at least substantially identical in the vaccinia virus prime/vaccinia virus boost regimen as compared to the DNA prime/vaccinia virus boost regimen as determined in "trial 1" and "trial 2" (preferably both) described in WO02/42480, the vaccinia virus is considered to induce at least substantially the same level of immunity in the vaccinia virus prime/vaccinia virus boost regimen as in the DNA prime/vaccinia virus boost regimen. More preferably, in at least one of the above assays, the CTL response after primary immunization with vaccinia virus/booster is higher than after DNA-primary immunization/booster with vaccinia virus. Most preferably, the CTL response is higher in both assays.

WO02/42480 discloses how to obtain vaccinia viruses having the above-mentioned properties of MVA-BN and derivatives thereof.

Methods for inserting the DNA defined above into poxvirus DNA, and methods for obtaining recombinant poxviruses are well known to those skilled in the art. In recombinant vaccinia viruses, expression of the DNA of the invention is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter, more preferably a vaccinia virus promoter. The DNA of the present invention is preferably inserted into a non-essential region of the viral genome. In another preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at the site of the natural deletion in the MVA genome (disclosed in PCT/EP 96/02926).

In summary, a most preferred embodiment of the invention provides a vector comprising a DNA as defined above, wherein said vector is MVA-BN or a derivative thereof, said DNA comprising an expression module encoding a flavivirus NS1 protein or a part thereof, preferably a dengue virus, more preferably dengue virus serotype 2.

In a preferred embodiment, the invention relates to the use of the NS1 protein or a part thereof encoded by the DNA of the invention, or the vector of the invention, for vaccination against multiple flaviviruses or flavivirus serotypes. For the definition of the NS1 protein or parts thereof according to the invention reference is made to the above-described section, wherein the DNA encoding NS1 has been defined as the product expressed by said DNA. The following summary of the proteins of the invention is therefore not to be construed as limiting the invention. In general terms, the NS1 protein may be an isolated NS1 protein encoded by any flavivirus or portion thereof. The NS1 protein or a portion thereof is preferably derived from dengue virus, most preferably dengue virus serotype 2. The protein may comprise only the amino acid sequence of the viral NS1 protein or a portion thereof. In a preferred embodiment, the NS1 protein may also contain additional amino acids necessary for efficient expression of the protein. Examples of such amino acid/amino acid sequences are shown above and include the methionine encoded by the appended ATG codon at the N-terminus of the protein and the amino acid sequence derived from the C-terminus of the E protein, the signal sequence required for glycosylating the NS1 protein or portions thereof. Other signal sequences are also within the scope of the present invention. In another embodiment, the NS1 amino acid sequence or portion thereof may be fused to other proteins/peptides. Examples of fusion partners are sequences allowing the recognition of the protein, such as a tag or other flavivirus protein or part thereof.

In a preferred embodiment, the invention relates to the DNA, vector or NS1 protein or parts thereof of the invention as a vaccine, in particular as a vaccine against multiple flaviviruses or flavivirus serotypes. A "vaccine" is a compound, i.e. DNA, protein, vector or virus, that induces a specific immune response.

According to an alternative to this embodiment, the "vaccine" according to the invention is based on a dengue virus NS1 protein or a part thereof capable of inducing an immune response against the NS1 protein of all serotypes of the dengue virus. In particular, it has been demonstrated that an immune response induced by one dengue virus serotype, in particular the NS1 protein of serotype 2, is resistant to the NS1 protein of at least two, preferably at least three, most preferably all serotypes of dengue virus, preferably also to at least one other mosquito-borne flavivirus.

As mentioned above, the present inventors have found that the NS1 protein or a portion thereof from one flavivirus of the present invention induces an immune response against the NS1 protein of another flavivirus. As indicated above, the "flavivirus" is preferably a mosquito-borne flavivirus. In other words, the inventors have found that in another embodiment, the NS1 protein or a portion thereof from one mosquito-borne flavivirus of the invention induces an immune response that is resistant to the NS1 protein of the mosquito-borne flavivirus from which the vaccine is derived and also resistant to the NS1 protein of other mosquito-borne flaviviruses. Thus, vaccines derived from mosquito-borne flaviviruses may serve as vaccines against one or more mosquito-borne flaviviruses. The term "vector derived from flavivirus" or similar terms in the context of this specification refers to the above-mentioned vector (e.g., poxvirus vector or plasmid) comprising the above-mentioned DNA. Thus, this term refers to the vector insert rather than the vector backbone. An example of a "flavivirus derived vector" is a poxvirus vector, such as MVA, comprising an expression module comprising a poxvirus promoter, a sequence encoding a flavivirus NS1 protein or part thereof preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and the coding sequence ending in a translation stop codon.

Thus, vaccination with the DNA, vector or NS1 protein or portions thereof can be as a single subunit vaccine against a wide range of flaviviruses or at least flaviviruses serotypes. DNA or vector encoding NS1 protein or a portion thereof from one flavivirus or flavivirus serotype, or NS1 protein or a portion thereof from said flavivirus or serotype, may thus be used as a vaccine against other flaviviruses and flavivirus serotypes by vaccination. For example, a vaccine derived from dengue virus serotype 2 may be used as a vaccine against one, two or all of serotypes 1, 3 and 4, as well as against serotype 2. It can further be used to protect individuals against other flaviviruses, such as west nile virus, japanese encephalitis virus, and yellow fever virus.

In a preferred embodiment, the DNA of the invention is used as a vaccine. It is known to the person skilled in the art that intramuscular injection of naked DNA, in particular DNA, containing a eukaryotic expression module according to the invention will lead to the expression of the protein encoded by the expression module. The protein is exposed to the immune system and elicits a specific immune response.

In another embodiment, the vaccination is performed by administering a vector according to the invention, in particular a viral vector, more preferably a poxvirus vector, most preferably a vaccinia virus vector, such as a MVA vector.

For the preparation of vaccines based on vaccinia virus, the virus according to the invention, in particular MVA-BN and derivatives thereof, can be converted into a physiologically acceptable form. This can be done based on the experience of preparing a poxvirus vaccine for the prophylactic vaccination of smallpox (see Stickl, H. et al [1974 ]]Dtsch.med.wscr.99, 2386-. For example, the purified virus was purified at 5x10⁸TCID₅₀The concentration of each/ml was prepared in about 10mM Tris, 140mM NaCl pH7.4, and stored at-80 ℃. For the preparation of vaccine injections (vaccine shots), for example, 10 is added²-10⁹The virus particles of (4) are lyophilized in 100ml Phosphate Buffered Saline (PBS) in an ampoule (preferably a glass ampoule) in the presence of 2% peptone and 1% human albumin.

Vaccines based on vaccinia virus, in particular based on MVA-BN, which are used for vaccination are particularly preferably stored in the lyophilized state. In the examples section it is shown that if the virus used for vaccination is stored in lyophilized form, the percentage of cross-reactivity induced by one flavivirus NS1 protein in the immune response and immune response to different flavivirus NS1 and flavivirus serotype NS1 proteins, respectively, will be very high. Thus, a bolus of vaccine is preferably produced by stepwise lyophilization of the virus in the formulation. Such formulations may contain additional additives suitable for in vivo administration, such as mannitol, dextran, sugar, glycine, lactose, or polyvinylpyrrolidone, or other additives, such as antioxidants or inert gases, stabilizers or recombinant proteins (e.g. human serum albumin). A typical formulation suitable for lyophilisation containing the virus contains 10mM Tris-buffer, 140mM NaCl, 18.9g/L dextran (MW 36000-. The glass ampoule is then sealed and stored at a temperature between 4 ℃ and room temperature for several months. However, it is preferred to keep the ampoule below-20 ℃ as long as it is not necessary to use it. For vaccination, the lyophilisate can be dissolved in 0.1-0.5ml of an aqueous solution, e.g., water, physiological saline or Tris buffer, administered by systemic or topical routes, i.e., by parenteral, intramuscular or any other route of administration known to those skilled in the art. The mode of administration, the dosage administered, and the number of administrations can be optimized by those skilled in the art by known means. Most preferably, the poxvirus vector is administered subcutaneously or intramuscularly. Most preferably, the vaccination is performed by two vaccine injections separated by, for example, 3-5 weeks.

If the vaccine is a MVA-BN vector or a derivative thereof comprising the DNA according to the invention, a particular embodiment of the invention relates to a vaccination kit comprising in a first bottle/container the MVA-BN viral vector according to the invention for the first vaccination (primary immunization) and in a second bottle/container the MVA-BN viral vector according to the invention for the second vaccination (booster immunization).

If the vaccine is a MVA-BN vector or derivative thereof containing the above DNA, a particular embodiment of the invention relates to the administration of the vaccine in a therapeutically effective amount in a first vaccination ("primary vaccination") and a second vaccination ("booster vaccination"). The time interval between the initial inoculation and the booster inoculation is, for example, 2 to 12 weeks, preferably 3 to 6 weeks, more preferably about 3 weeks. The virus amount for inoculation and injection is at least 1x10²TCID₅₀Preferably 1x10⁷TCID₅₀To 1x10⁹TCID₅₀. Furthermore, a particular embodiment of the present invention relates to an vaccination kit comprising in a first bottle/container the above-mentioned MVA-BN viral vector for a first vaccination (primary immunization) and in a second bottle/container the above-mentioned MVA-BN viral vector for a second vaccination (booster immunization).

Thus, in a vaccine embodiment, the invention relates to a vaccine comprising the DNA, vector or NS1 protein or a portion thereof as described above, and the use of the DNA, vector or protein for the preparation of a vaccine. According to a preferred embodiment, the invention relates to the use of said DNA, vector or protein for the preparation of a vaccine, wherein the NS1 protein or a part thereof, the NS1 protein encoded by the DNA or vector or a part thereof is from one dengue virus serotype, and wherein the DNA, vector or NS1 protein or a part thereof is used as a vaccine against two, three or all dengue virus serotypes. Most preferably, the dengue virus serotype is serotype 2.

The present invention further relates to a method for treating or preventing a flavivirus infection comprising inoculating an animal (including a human) in need thereof with the above DNA, the above vector, or the above NS1 protein or a portion thereof. In particular, the present invention relates to the above method, wherein the NS1 protein or a part thereof, or the NS1 protein encoded by the DNA or vector or a part thereof, is from a dengue virus serotype, wherein the DNA, vector or NS1 protein or part thereof acts as a vaccine against two, three or all serotypes of the dengue virus.

Summary of The Invention

In particular, the invention relates to the following, alone or in combination:

the use of a substance in the preparation of a vaccine,

-a nucleic acid comprising an expression component comprising a transcriptional regulatory element and a sequence encoding at least the NS1 protein of a mosquito-borne flavivirus or a part thereof,

-a vector comprising said nucleic acid, and/or

-the NS1 protein of said flavivirus or a part thereof,

the vaccine is useful against a mosquito-borne flavivirus that produces the nucleic acid or produces the NS1 protein or portion thereof, and against at least one other mosquito-borne flavivirus.

The use as described above, wherein the mosquito-borne flavivirus from which said nucleic acid is derived or from which said NS1 protein or portion thereof is derived is dengue virus.

The use of a substance in the preparation of a vaccine,

-a nucleic acid comprising an expression module containing transcriptional regulatory elements and at least a sequence encoding the NS1 protein of the dengue virus serotype or a part thereof,

-a vector comprising said nucleic acid, and/or

-the NS1 protein of said dengue virus serotype or a part thereof,

the vaccine is useful against all serotypes of dengue virus and may be selected against at least one other mosquito-borne flavivirus.

The use as described above, wherein the dengue virus that produces said nucleic acid or that produces said NS1 protein or a part thereof is dengue virus serotype 2.

The use as described above, wherein the sequence encoding the NS1 protein of a mosquito-borne flavivirus or dengue virus serotype, or a part thereof, is preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and wherein the coding sequence terminates at a translation stop codon.

The use as described above, wherein said other mosquito-borne flavivirus is selected from the group consisting of West Nile virus, yellow fever virus and Japanese encephalitis virus.

The use as described above, wherein the vector is a poxvirus vector.

The use as described above, wherein the poxvirus vector is a modified vaccinia virus ankara (MVA) strain, in particular MVA-BN deposited at the european collection of animal cells under number V00083008 or a derivative thereof.

The use as described above, wherein said poxvirus vector has been lyophilized and can be reconstituted in a pharmaceutically acceptable diluent prior to administration.

The use as described above, wherein the transcriptional regulatory element is a poxvirus promoter.

The use as described above, wherein the vaccine is administered in a therapeutically effective amount in a first vaccination ("primary vaccination") and a second vaccination ("booster vaccination").

A method for the treatment or prevention of a flavivirus infection comprising vaccinating an animal, including a human, in need thereof

-a nucleic acid comprising an expression component comprising a transcriptional regulatory element and at least a sequence encoding a mosquito-borne flavivirus NS1 protein or a part thereof,

-a vector comprising said nucleic acid, and/or

-the NS1 protein of said flavivirus or a part thereof

Wherein the flavivirus infection is an infection by a mosquito-borne flavivirus that produces the nucleic acid or that produces the NS1 protein or portion thereof, and/or an infection by another mosquito-borne flavivirus.

The method as above, wherein the mosquito-borne flavivirus that produces the nucleic acid or that produces the NS1 protein or portion thereof is dengue virus.

A method for the treatment or prevention of a flavivirus infection comprising vaccinating an animal, including a human, in need thereof

-a nucleic acid comprising an expression module comprising transcriptional regulatory elements and at least a sequence encoding a dengue virus serotype NS1 protein or a part thereof,

-a vector comprising said nucleic acid, and/or

-the NS1 protein of said dengue virus serotype or a part thereof,

wherein the flavivirus infection is by a dengue virus serotype that produces the nucleic acid or produces the NS1 protein or portion thereof, and/or by other dengue virus serotypes, and/or by other mosquito-borne flaviviruses.

The method above, wherein the dengue virus that produces the nucleic acid or that produces the NS1 protein or portion thereof is dengue virus serotype 2.

The above method wherein the sequence encoding the NS1 protein of a mosquito-borne flavivirus or dengue virus serotype, or a portion thereof, is preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and wherein the coding sequence terminates at a translation stop codon.

The method above, wherein the vector is a poxvirus vector.

The method above, wherein the poxvirus vector is a modified vaccinia virus ankara (MVA) strain.

The above method, wherein the MVA strain is MVA-BN deposited at the European Collection of animal cells under the number V00083008 or a derivative thereof.

The method wherein said poxvirus vector has been lyophilized and reconstituted in a pharmaceutically acceptable diluent prior to administration.

The method above, wherein the transcriptional regulatory element is a poxvirus promoter.

The method as above, wherein the poxvirus vector or pharmaceutical composition is administered in a therapeutically effective amount in a first inoculation ("primary inoculation") and in a second inoculation ("booster inoculation").

A poxvirus vector carrying DNA comprising an expression module comprising a transcriptional regulatory element and at least a sequence encoding a flavivirus NS1 protein or a part thereof, wherein the poxvirus is modified vaccinia virus ankara (MVA) strain BN or a derivative thereof deposited at the european collection of animal cells under the number V00083008.

The poxvirus vector of any one of the preceding claims, wherein the flavivirus is a mosquito-borne flavivirus, in particular a dengue virus.

The poxvirus vector of any preceding claim, wherein said dengue virus is dengue virus serotype 2.

The poxvirus vector wherein the sequence encoding the flavivirus NS1 protein, or portion thereof, is preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and wherein the coding sequence terminates at a translation stop codon.

The poxvirus vector of any one of the preceding claims, wherein the transcriptional regulatory element is a poxvirus promoter.

The poxvirus vector of any one of the preceding claims, wherein the poxvirus vector is lyophilized.

The poxvirus vector as described above as a vaccine.

A pharmaceutical composition comprising the poxvirus vector as defined above and a pharmaceutically acceptable carrier (carrier), diluent and/or additive.

The poxvirus vector or the pharmaceutical composition as defined above for use in the treatment and/or prevention of a flavivirus infection, wherein the poxvirus vector or the pharmaceutical composition is administered in a therapeutically effective amount in a first inoculation ("primary inoculation") and in a second inoculation ("booster inoculation").

A method for the treatment and/or prevention of flavivirus infection comprising vaccinating an animal, including a human, in need thereof with said vector or said pharmaceutical composition.

A cell, preferably a human cell, comprising the poxvirus vector as described above.

Use of the poxvirus as defined above for the preparation of a vaccine for the treatment or prevention of a flavivirus infection.

A kit for primary immunization/booster immunization comprising in a first bottle/container said poxvirus vector or said pharmaceutical composition for primary vaccination (primary immunization) and in a second bottle/container said poxvirus vector or said pharmaceutical composition for secondary vaccination (booster immunization).

Drawings

FIG. 1A: the "signal sequence + NS 1" cDNA protein coding sequence of dengue NGC strain of the construct of the invention is an example. The natural start site of the NS1 gene is indicated by an arrow. The important feature is the addition of an ATG start codon and a stop codon ("TAG" in this case). Nucleotide sequence numbers refer to positions in the genome of the NGC strain (Genbank accession No. AF 038403). The nucleotide and amino acid sequences in figure 1A correspond to SEQ: ID No. 5. Amino acids are shown individually in SEQ: ID No. 6.

FIG. 1B: map of plasmid pAF7NS1 containing the "signal sequence + NS 1" protein coding sequence of dengue NGC strain.

FIG. 1C: the nucleotide sequence of the NS1 module in plasmid pAF7 shows the primer binding sites located on this module when PCR amplified with oBN345 and oBN 338. The nucleotide and amino acid sequences in figure 1C correspond to SEQ: ID No. 7. Amino acids are shown individually in SEQ: ID No. 8.

FIG. 1D:upper drawing: Kyte-Doolittle hydrophilicity profile of the amino acid sequence of dengue NGC strain NS1 (amino acid 776-1127 of dengue NCG polyprotein Genbank accession AF 038403). Values above zero are hydrophobic.Lower view of: Kyte-Doolittle hydrophilic map of the amino acid sequence of the dengue NGC strain NS1, which contains a signal sequence derived from the last 28 amino acids of the C-terminus of the E protein (amino acids 748-775). The complete amino acid sequence represents amino acids 748-1127 of dengue NGC polyprotein (Genbank accession AF038403), which for this strain starts with the "ATG" start codon but lacks a stop codon. Sig is the signal sequence. Values above zero are hydrophobic.

FIG. 2: nucleotide sequence of the expression module "poxvirus promoter + signal sequence + NS 1". The nucleotide and amino acid sequences in figure 2 correspond to SEQ: ID No. 9. Amino acids are shown individually in SEQ: ID No. 10. Briefly, the minimal early/late promoter element of poxvirus controls the expression of NS1 protein of dengue virus serotype 2, where the N-terminus of NS1 protein is fused to the 28C-terminal amino acids of the E protein. Translation is terminated at a TAG stop codon inserted into the nucleic acid sequence.

FIG. 3A: the NS1 expression module was cloned into the XhoI site at the blunt end of pBNX07 (blunt end cloning) to yield clone pBN 41. PPr ═ poxvirus promoter, D2F1 ═ 1 flanking deletion site 2, NPT II ═ neomycin resistance gene, IRES ═ internal ribosome binding site, EGFP ═ enhanced green fluorescent protein, NS1 (in pBN 41) ═ signal sequence + NS1, D2F2 ═ 2 flanking deletion site 2, Sig ═ signal sequence. AmpR ═ ampicillin resistance gene.

FIG. 3B: the HindIII map of MVA (Genbank U94848), shows the position of the six deletion sites (-J- ═ linker of the deletion site) of MVA. The "PPr + NPT II + IRES + EGFP + PPr + NS 1" module was inserted into deletion site 2 of MVA. PPr ═ poxvirus promoter, NPT II ═ neomycin resistance gene (protein coding sequence), IRES ═ internal ribosome binding site, NS1 ═ signal sequence + NS1 protein coding sequence of dengue 2NGC strain.

FIG. 4: ELISA absorbance profile showing serum titration after immunization of all three rabbits.

FIG. 5: ELISA cross-reactivity study. Cross-reactivity of rabbit sera at day 38 (upper) and day 66 (lower) with lysates from cells that had been infected with DENV-1, DENV-3, DENV-4, JEV and WNV in ELISA-assays.

Examples

The following examples further illustrate the invention in detail. It should be understood by those skilled in the art that the following examples are not to be construed as limiting the application of the present technology to these examples in any way.

Example 1: mBN07 construction

Details of NS1 antigen (FIG. 1)

This example relates to NS1 derived from serotype 2 of the New Guinea C strain-NGC strain (e.g., Genbank sequence AF 038403). Since the NS1 protein of flavivirus is produced as part of a polyprotein precursor, the NS1 gene in the corresponding DNA is not preceded by an "ATG" start codon.

Therefore, the cDNA sequence encoding the NS1 protein must have an "ATG" initiation codon added. The addition of a signal sequence then glycosylated the newly synthesized NS1 protein within the endoplasmic reticulum. Finally, the protein coding module requires a stop codon, in this example TAG, added to the 3' end of the protein coding cDNA sequence. In the examples used herein, the "ATG + signal sequence" element is derived from the hydrophobic C-terminus (the last 28 amino acids, where the NGC strain starts with amino acid m (ATG)) of the E protein.

FIG. 1A shows the exact signal sequence used as an example of the invention plus the sequence of NS1 (see SEQ: ID5 and 6). The "signal sequence + NS 1" nucleotide coding sequence was obtained from dengue NGC genomic RNA by RT-PCR amplification using the following primers:

upstream of D2NS 1-1: 5' -ACAGGAATGAATTCACGTAGCACCTCA-3′(SEQ：ID NO 4)

The Bgl II restriction endonuclease recognition site is in italics.

The start codon is underlined.

Downstream of D2NS 1-2: 5' -AATCTACTAGGCTGTGACCAAGGAGTT-3′(SEQ：ID NO 3)

The Bgl II restriction endonuclease recognition site is in italics.

The stop codon is underlined.

RT-PCR amplification was performed using the Titan One Tube RT-PCR kit from Roche Molecular Biochemical (Catalog No.1-939-823) according to the manufacturer's recommendations. However, virtually any commercial or non-commercial RT-PCR kit may be used.

The RT-PCR product can then be cloned into the BamHI site of any of the multiple cloning sites in many commercially available bacterial cloning plasmids, but in this example it is cloned into pAF7, resulting in the clone pAF7D2NS 1-the sequence of which is detailed in FIGS. 1B and 1C. FIG. 1D shows a hydrophobic map of NS1 amino acid sequence and NS1 with the addition of a signal sequence in the C-terminal amino acid coding sequence of the E protein.

Details of NS1 expression modules (FIG. 2)

To express the "signal sequence + NSI" from a poxvirus vector such as canarypox, fowlpox, vaccinia or MVA, a poxvirus promoter is added to the 5' end of the cDNA. Polyadenylation signal sequence is not necessary as all poxvirus synthetic RNAs are polyadenylated by the virus-encoded enzyme, which does not require a polyA additional signal sequence to perform this function. Any poxvirus promoter may be used for expression of this component. FIG. 2 and SEQ: ID Nos. 9 and 10 show the nucleotide sequences of the "poxvirus promoter + signal sequence + NS 1" module as an example of the invention.

As an example used in the present invention, "signal sequence + NS 1" was further PCR amplified from NS1 plasmid clone with primers oBN338 and oBN 345. oBN345 primer contains the nucleic acid sequence of the poxvirus minimal promoter element 5' of the target sequence in the cloned plasmid. oBN345 the plasmid target sequence required for primer binding is about 40 nucleotides upstream of the start codon of the signal sequence. This ensures that the RNA transcript contains a non-protein coding sequence in front of the ATG start codon of the signal sequence.

PCR primers and Ps promoter:

oBN338：5′-TTGTTAGCAGCCGGATCGTAGACTTAATTA(30mer)(SEQ：ID No.1)

oBN345：

5′-CAAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAACACGATAATACCATGG-3′

(SEQ：ID No.2)

(underlined nucleotides represent poxvirus minimal promoter sequences.)

The annealing temperature of 5 cycles prior to the PCR amplification reaction was calculated from the nucleotide sequence that bound to the homologous sequence in the oBN34 cloning vector.

Integration of NS1 expression modules into MVA (FIG. 3)

The PCR amplification product was blunt-ended and cloned into the nicked and blunt-ended XhoI site of plasmid pBNX07 (see FIG. 3a) to form plasmid pBN41 (see FIG. 3 a). pBN41 is a vector that integrates the "pox promoter + signal sequence + NS 1" component into deletion site 2 of MVA by homologous recombination.

The basic features of pBN41 (fig. 3a) are as follows:

the plasmid backbone is pBluescript SK from Stratagene⁺(Genbank VB0078)

-D2F 1: deletion site 2 is flanked by 1 homologous recombination arms. It represents the nucleotide sequence 20117 to 20717 of the MVA Genbank sequence U94848.

-PPr: a poxvirus promoter.

-NPT II: neomycin phosphotransferase protein coding sequence (protein coding sequence of Genbank V00618).

-IRES: internal ribosome entry sequences from encephalomyocarditis virus (Jang et al, 1989, Genbank M16802).

-EGFP: enhancing the coding sequence of Green fluorescent protein (protein coding sequence-nucleotide 675 to nucleotide 1394 of Genbank sequence U57609)

-NS 1: the "signal sequence + NS 1" protein coding sequence from dengue NGC strain.

-D2F 2: deletion advantage 2 flanking 2 homologous recombination arms. It represents the nucleotide sequence 20719 to 21343 of the MVA Genbank sequence U94848.

-AmpR: ampicillin resistance gene of pBluescript

3.1 insertion of dengue "pox promoter + Signal sequence + NS 1" into M by homologous recombinationDeletion site of VA

3.1.1 integration into the MVA genome by homologous recombination

The integration vector pBN41 described above integrated the dengue NS1 expression module plus a reporter module (pox promoter + NPT II-IRES-EGFP) into the MVA genome by homologous recombination between flanking 1 and flanking 2 arms of pBN41 and the homologous target sequences in the MVA genome. It is accomplished by transfecting a linear integration vector into Chick Embryo Fibroblasts (CEF) that have been infected with a MVA of low multiplicity of infection (MOI, e.g., 0.01 infectious units per cell). At 48 hours post-infection or when the infection reaches confluence, viral extracts are prepared and stored at-20 ℃ in preparation for screening and clonal purification of the desired recombinant mva (rmva).

3.1.2 screening and clonal purification of rMVA

Elimination of non-recombinant MVA (empty vector Virus) and amplification of rMVA in the presence of G418 (the amount of G418 must be optimized in order to determine the highest dose that does not kill CEF cells)Is low inMOI was achieved by infection of confluent Chicken Embryo Fibroblasts (CEF). Any virus that does not contain or integrate the NPT2 gene will not replicate when G418 is added to the cell maintenance medium. G418 inhibits DNA replication, but since CEF cells will be in a stable non-replicating stage, CEF cells will not be affected by the action of G418. Due to the enhanced expression of fluorescent green protein, rfva infected CEF cells can be observed under a fluorescent microscope.

Viral extracts from the homologous recombination step must be serially diluted and used to infect fresh CEF cells in the presence of G418 and covered with low melting agarose. After 2 days of infection, agarose-infected plates were observed under a fluorescent microscope, and only green infected cells were observed. These cells are labeled and the agarose containing the foci of cell infection can be removed and placed into a small 1.5ml centrifuge tube containing sterile cell maintenance medium. The virus was released from the agarose plug by freeze-thawing a small centrifuge tube three times at-20 ℃.

The best clones were further clone purified under agarose until no signs of empty vector contamination were present by PCR analysis (3 to 30 clone purification cycles). These clones were then amplified by RT-PCR to further determine rigorously whether the correct insertion configuration was obtained, confirm the sequence of the promoter-foreign gene assembly and analyze expression. After these analyses, only one clone was further amplified under G418 selection conditions in order to prepare a mother liquor for further identification and immunogenicity studies.

The recombinant MVA inserted with the dengue NS1 expression module of the invention was named mBN 07. FIG. 3b shows the configuration of the exogenous sequence inserted in mBN 07.

4. MVA expression of authentic NS1

Expression of NS1 protein from recombinant MVA, mBN07, was confirmed by standard western blot analysis under non-denaturing conditions. More specifically, mammalian tissue culture cells, such as BHK-21 cells, were infected with purified mBN07 having an MOI of 1.0 infectious unit/cell and analyzed for NS1 expression. Crude protein extracts were prepared from those infected cells 24-30 hours after infection, and a portion was mixed with SDS-PAGE gel loading buffer containing 2 mercaptoethanol (2-ME) or no 2-ME. These samples, plus a protein extract from cells infected with dengue NGC strain (mosquito cell line) as a positive control, were electrophoretically separated in SDS-PAGE gels and then blotted onto nitrocellulose membranes. The membrane was probed with an anti-dengue NS1 monoclonal antibody.

The results show that NS1 expressed in mBN07 was recognized by the anti-dengue NS1 monoclonal antibody and formed a correct dimeric form similar to NS1 from dengue infected cells (compared to the unboiled mBN07 lane without 2-ME and the unboiled DEN2 lane without 2-ME). In addition, the dimeric form was shown to decompose to the monomeric form under denaturing conditions (see boiling mBN07 lane containing 2-ME).

In western blot analysis, NS1 expressed in cells infected with mBN07 was also recognized by pooled convalescent patient sera and monoclonal antibodies that cross-react with NS1 of all four dengue serotypes. This indicates that mBN 07-expressed NS1 is immunogenic.

Throughout the examples section mBN07 (sometimes also referred to as BN07) was stored in liquid (optionally frozen) or lyophilized state. To obtain a lyophilized virus, a solution containing the virus was prepared, which contained 10mM Tris-buffer, 140mM NaCl, 18.9g/L dextran (molecular weight 36000-40000), 45g/L sucrose, 0.108g/L monopotassium L-glutamate monohydrate pH 7.4. The formulation was then lyophilized. For reconstitution, water is added to the lyophilized formulation.

Example 2: mBN07 Cross-immunogenicity of NS1 against dengue viruses NS1 and Japanese Encephalitis Virus (JEV) NS1 and West Nile Virus (WNV) NS1, except serotype 2

1.NS1 expressed from mBN 07: reactivity to serum of convalescent patients

Detecting the possibility that NS1 expressed in mBN07 was recognized by sera of convalescent patients from individuals with prior dengue virus infection. Sera of 68 individuals with antibodies against the envelope protein of dengue virus, which were confirmed by immunoblotting to be resistant to the authentic antigens of dengue serotypes 1 to 4, were taken and the immunoblot strips prepared from the mBN07 infected cell extract were examined, with the extract of MVA-GFP infected cells being used as a control. The antigen-containing cell lysate was treated with loading buffer without 2-mercaptoethanol and without heating. In the serum of 68 individuals tested, 62 (91.2%) reacted with BN07 NS1 expressed by mBN07 in the immunoblot. These sera were further analyzed for reactivity to NS1 of all 4 dengue virus serotypes and Japanese Encephalitis Virus (JEV). The results are shown in Table 1. 54 parts of serum were reacted with NS1 of all serotypes of dengue virus and Japanese Encephalitis Virus (JEV), and 53 parts (98.2%) of the 54 parts of serum were also reacted with mBN 07-expressed NS 1. The 7 sera were specific for NS1 of at least one dengue virus serotype and did not react with NS1 of JEV. All 7 sera also reacted with mBN 07-expressed NS 1. An additional 7 sera reacted only with NS1 of JEV and not with NS1 of any dengue virus serotype, whereas 2 of these JEV-specific sera (28.6%) also reacted with NS1 expressed in mBN 07.

TABLE 1

	BN07 NS1 negative	BN07 NS1 positive
			True DEN NS1 positive	0％(0/7)	100％(7/7)
True DEN + JEV NS1 positivity	1.85％(1/54)	98.15％(53/54)
			True JEV NS1 positivity	71.43％(5/7)	28.57％(2/7)

Comparison of the antisera response of NS1 expressed by authentic NS1 and mBN 07. In the parentheses: the number of positive detection samples/the total number of detection samples. DEN-dengue and JEV-japanese encephalitis virus.

The same 68 sera were also analyzed for reactivity against pre-membrane proteins. In the inventors' experience, antibodies against the pre-membrane were more specific than antibodies against NS1 or E. Thus, a patient infected with dengue will develop antibodies that recognize the dengue virus pre-membrane rather than the JEV pre-membrane, and vice versa. Analysis along this line will provide a better prediction of the individual's history of infection. Table 2 shows that sera from 22 patients reacted only with authentic dengue pre-membrane protein, suggesting that these 22 patients were exposed only to dengue virus and not to JEV. All 22 sera reacted with mBN 07-expressed NS 1. An additional 22 patients had evidence of prior infection with dengue fever and JEV, and they also all reacted with NS1 expressed at mBN 07. There were also 21 patients in this group who had previous evidence of infection with JEV alone (although these sera had cross-reactive antibodies against dengue E). Interestingly, 17 of these 21 JEV responders (82%) reacted with mBN 07-expressed NS 1. Of the entire group, only 3 sera were not reactive with dengue or JEV's pre-membrane protein, of which only 1 reacted with mBN 07-expressed NS 1. The most likely reason for this is that the antibody titer is too low to be detected by immunoblotting.

TABLE 2

	BN07 NS1 negative	BN07 NS1 positive
			True DEN prM positivity	0％(0/22)	100％(22/22)
True DEN + JEV prM positivity	0％(0/22)	100％(22/22)
			True JEV prM positivity	19.0％(4/21)	81.0％(17/21)
PrM negative	66.7％(2/3)	33.3％(1/3)

Antisera responses against authentic pre-membrane and BN07 NS1 were compared. In the parentheses: the number of positive detection samples/the total number of detection samples. DEN-dengue and JEV-japanese encephalitis virus.

The data in table 2 also clearly show that of the 6 sera that did not react with NS1 expressed at mBN07, 4 were from individuals previously infected with JEV but not dengue, and the remaining 2 were free of detectable antibodies to dengue or JEV pre-membrane proteins, probably due to low titers.

2. Rabbit was inoculated with mBN07 and post-immune sera to dengue virus and Japanese encephalitis virus detected by immunoblot and ELISA analysis

Three rabbits without specific pathogens were immunized subcutaneously following the vaccination program shown below. Each rabbit was vaccinated on days 0 and 28 with a vial of lyophilized vaccine (1x10e8 TCID50 BN07 lyophilized vaccine) reconstituted to 1ml with sterile water. Blood samples were taken before the first inoculation (pre-blood withdrawal) and 10 days after the second inoculation.

Day 0-first vaccination after blood pre-withdrawal

Second inoculation on day 28

On day 38, blood was collected

Third inoculation on day 56

On day 66, blood was collected

On day 112, 50ml of blood was drawn from each rabbit

2.1Pre-bleed and post-immune serum detection against dengue serotype 2 immunoblots

Dengue 2 virus antigen and antigen from uninfected C6/36 cells were separated by SDS PAGE under non-denaturing conditions. For immunoblot analysis, day 38 sera (diluted 1: 200) were used.

The results clearly show that vaccination mBN07 resulted in high titers of anti-NS 1 antibody in all three rabbits, and that this antibody cross-reacted with authentic NS1 produced after infection of tissue culture mosquito cells with dengue serotype 2. Serum collected prior to vaccination did not react with any dengue protein on the immunoblot.

Post-immunization sera at day 38 at 1: 1000, 1: 2000, 1: 4000, 1: 10^-4，1∶10^-5，1∶10^-6The samples were titrated down and examined under non-denaturing conditions on immunoblot strips of dengue 2 virus antigen and control strips of uninfected C6/36 cells separated by SDS PAGE. The endpoint titers of the three rabbit sera at day 38 were calculated to be 1: 10,000. The serum endpoint titer at day 66 was calculated as 1x10 in both the immunoblot and ELISA assays⁵(data not shown).

Serum before and after immunization was 1: 10^-2，1∶10^-3，1∶10^-4，1∶10^-5，1∶10^-6，1∶10^-7Titrated in time and detected in an indirect IgG ELISA. Wells were coated with 1: 250 dilution of dengue 2 and uninfected C6/36 lysate.

TABLE 3

ELISA Absorbance at different dilutions was shown for Pre-and Post-immune sera from each rabbit (Pre ═ Pre-immune sera, Post ═ Post-immune sera)

Post-immunization serum titrations of each rabbit are shown in figure 4. Post-immunization sera from each rabbit were estimated at an endpoint dilution of 1: 1000.

2.2Pre-bleed and post-immune serum detection against dengue serotype 1, 3, 4 and Japanese encephalitis and West Nile virus immunoblots

Day 38 sera from each rabbit were tested at a 1: 1000 dilution on immunoblot strips of dengue 1, 2, 3, 4 and JE virus antigens separated by SDS PAGE under non-denaturing conditions plus a control strip of uninfected C6/36 cells. The post-immunization sera of each rabbit were shown to react with NS1 from dengue serotypes 1, 3 and 4 and with NS1 on japanese encephalitis immunoblots.

Day 66 each rabbit serum was tested at a 1: 1000 dilution on immunoblot strips of dengue 1, 3, 4, WNV and JE viral antigens separated by SDS PAGE under non-denaturing conditions plus a control strip of uninfected C6/36 cells. The post-immunization sera of each rabbit were shown to react with NS1 from dengue serotypes 1, 3 and 4 and with NS1 on immunoblots of japanese encephalitis virus and west nile virus.

To confirm the immunoblot analysis, an Elisa cross-reaction analysis was performed. Wells on microtiter plates were coated with 1: 250 dilutions of lysates of DENV-1, DENV-3, DENV-4, JEV, WNV and uninfected cells. The sera were the sera at day 38 (fig. 5A) and day 66 (fig. 5B).

From the immunoblot analysis and ELISA assays it can be concluded that: antibodies induced by dengue virus NS1 cross-react with all other dengue serotypes, JEV and WNV.

2.3. Conclusion

Rabbits immunized with the mBN07 vaccine induced antibodies recognizing the true dengue virus serotype 2NS 1.

When the endpoints in immunoblot analysis and ELISA were 1: 10, respectively^-4And 1: 10^-3A very high immune response was observed.

Antibodies induced in rabbits cross-react with all other dengue serotypes (1, 3 and 4).

The antibody also cross-reacts with NS1 from heterologous viruses, such as JEV and WNV.

3. Immunogenicity studies in mice

Female distant mice were immunized by intraperitoneal route with mBN07 expressing dengue virus NS1 in varying amounts and schedules as shown below. mBN08 (a MVA corresponding to mBN07 but not expressing NS 1) and PBS served as controls. Mouse sera were used to test whether antibodies produced in mice were able to react on Western blots with NS1 protein from different flavivirus serotypes and japanese encephalitis virus, respectively. Sera from control mice were negative in all experiments.

The following groups were analyzed:

in the immunoblot assay, the following results were obtained:

TABLE 7

Very similar experiments were obtained with Balb/c mice: mice were vaccinated twice with 1x10 on days 0 and 21⁸TCID₅₀BN07 (lyophilized and reconstituted with water). Serum was collected on day 42. 100% of the sera were reactive with dengue virus 2NS1 and 100% of the sera were reactive with dengue virus from all four dengue virusesThe NS1 protein of the virus serotype reacted, and 75% of the sera reacted with the NS1 protein of JEV. With 1x10⁸TCID₅₀The results of non-lyophilized BN07 vaccination were as follows: 100% of the sera reacted with NS1 protein from all four dengue virus serotypes. Sera recognizing NS1 from JEV were inferior to sera obtained from mice inoculated with lyophilized BN 07.

And (4) conclusion:

antibodies with a very strong response to DENV-2 NS1 in 100% of mice immunized with BN 07.

The immune response of mice immunized with 1x10e7 TCID50 BN07 was as strong as the immune response of mice immunized with 1x10e8 TCID50 BN07 (data not shown).

The best percent cross-reactivity was observed in mice immunized 4 weeks apart and 4 weeks prior to blood draw.

Mice immunized with the lyophilized vaccine of BN07 had a stronger response to NS1 than mice immunized with the non-lyophilized vaccine.

Reference to the literature

Nimmannitya S，Kalayanaroo S，Nisalak A，andInnes B.1990.Second attack of dengue hemorrhagic fever.Southeast Asian Journal of Tropical Medicine and Public Health，21：699

Burke DS and Monath TP.，2001，Flaviviruses.In Fields Virology，Fourth Edition，Edited by David M Knipe and Peter M Howley.Published by Lippincott Williams andWilkins，Philadelphia.Pages 1043-1125.

Flamand M，Megret F，Mathieu M，LePault，ReyFA and Deubel V.，1999.Dengue Virus Type 1Nonstructural Glycoprotein NS1 is Secreted from mammalian cells as a soluble hexamer in a glycsylation-dependent fashion.J.Virol.，73：6104-6110.

Jang SK，Davies MV，Kaufman RJ and Wimmer E.，1989.Initiation of protein synthesis by internal entry of ribosomes into the5′nontransfated region of encephalomycarditis virus RNA in vivo.J.Virol.，63：1651-60.

Lindenbach BD and Rice CM.，2001，Flaviviruses and their replication.In Fields Virology，Fourth Edition.Edited by David M Knipe and Peter M Howley.Published by Lippincott Williams and Wilkins，Philadelphia.Pages 991-1041.

Schlesinger JJ，Brandriss MW and Walsh EE.，1987.Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural protein NS1.J.Gen.Virol.，68：853-7

Schesinger JJ，Foltzer M and Chapman S.，1993.The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against yellow fever encephalitis in mice.Virology.192：132-41

The techniques and procedures described in this specification are familiar to those skilled in the art of molecular biology and virology, particularly flavivirus virology and poxvirus genetic engineering. The techniques and procedures can be found in more detail in the following documents:

Molecular Cloning，A laboratory Manual.Second Edition.By J.Sambrook，E.F.Fritsch and T.Maniatis.Cold Spring Harbor Laboratory Press.1989.

Virology Methods Manual.Edited by Brian WJ Mahy and Hillar O Kangro.Academic Press.1996.

Molecular Virology：A Practical Approach.Edited by AJ Davison and RM Elliott.The Practical Approach Series.IRL Press at Oxford University Press.Oxford 1993.Chapter 9：Expression of genes by vaccinia virus vectors.

Current Protocols in Molecular Biology.Publisher：John Wiley and SonInc.1998.Chapter 16，sectionIV：Expression of proteins in mammalian cells using vaccinia viral vector.

Antibodies，A Laboratory Manual.By Ed Harlow and David Lane.Cold Spring Harbor Laboratory Press.1988.

instructions for the preservation of microorganisms

(the second of the design reside in 13)

Instructions for the preservation of microorganisms

(the second of the design reside in 13)

Claims (51)

1. A poxvirus vector carrying DNA comprising an expression module comprising a transcriptional regulatory element and a shorter epitope chain encoding the complete flavivirus NS1 protein or which still elicits an immune response, wherein the poxvirus from which the poxvirus vector is derived is the modified vaccinia virus ankara (MVA) BN strain deposited at the european collection of cell cultures under the number V00083008.

2. Poxvirus vector according to claim 1, wherein the expression module further comprises a sequence encoding a further flavivirus peptide/protein, which comprises less than 40 amino acids, if derived from the E protein, but at least a glycosylation signal sequence.

3. The poxvirus vector of claim 1 or 2, wherein the flavivirus is a mosquito-borne flavivirus.

4. The poxvirus vector of claim 3, wherein the mosquito-borne flavivirus is dengue virus.

5. The poxvirus vector of claim 4, wherein the dengue virus is dengue virus serotype 2.

6. Poxvirus vector according to anyone of claims 1 to 5, wherein the sequence encoding the flavivirus NS1 protein or a shorter epitope chain thereof still capable of eliciting an immune response is preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and wherein the coding sequence ends at a translation stop codon.

7. Poxvirus vector according to anyone of claims 1 to 6, wherein the transcriptional regulatory element is a poxvirus promoter.

8. Poxvirus vector according to anyone of claims 1 to 7, wherein the poxvirus vector is lyophilized.

9. A pharmaceutical composition comprising a poxvirus vector according to anyone of claims 1 to 8 and a pharmaceutically acceptable carrier, diluent and/or additive.

10. Poxvirus vector according to anyone of claims 1 to 8 as a vaccine.

11. The poxvirus vector of claim 10 wherein the vaccine is used to vaccinate against the flavivirus or flavivirus serotype producing the nucleic acid or producing the NS1 protein or its shorter epitope chain that is still capable of eliciting an immune response, and/or against one or more other flaviviruses and/or flavivirus serotypes.

12. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcriptional regulatory element and encoding an intact flavivirus NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response in the preparation of a vaccine against a flavivirus or flavivirus serotype that produces the NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, and/or against one or more other flaviviruses and/or flavivirus serotypes.

13. The use of claim 12, wherein the expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids but at least a glycosylation signal sequence.

14. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcriptional regulatory element and encoding an intact flavivirus NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, in the manufacture of a medicament for the treatment or prevention of a flavivirus infection in an animal, including a human, in need thereof, wherein the flavivirus infection is an infection of a flavivirus or flavivirus serotype that produces the nucleic acid or produces the NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, and/or an infection of another flavivirus and/or flavivirus serotype.

15. The use of claim 14, wherein the expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids but at least a glycosylation signal sequence.

16. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcription regulatory element and a shorter epitope chain encoding the complete flavivirus NS1 protein or which is still capable of eliciting an immune response in the preparation of a medicament for the prevention of infection by multiple flaviviruses or flavivirus sera in an animal, including a human, in need thereof.

17. The use of claim 16, wherein the expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids but at least a glycosylation signal sequence.

18. Use according to any one of claims 12 to 17, wherein the poxvirus vector is a poxvirus vector according to one of claims 1 to 8.

19. Poxvirus vector according to claim 11, for use according to anyone of claims 12 to 17, wherein the NS1 protein or its shorter epitope chain still capable of eliciting an immune response is derived from one dengue virus serotype, and wherein the vaccine protects an individual against infection by at least two dengue virus serotypes.

20. The poxvirus vector or use of claim 19, wherein the vaccine protects an individual against infection by all serotypes of dengue virus.

21. The poxvirus vector or use of any one of claims 19 to 20, wherein the dengue virus serotype and/or flavivirus from which the NS1 protein is produced is dengue virus serotype 2.

22. The poxvirus vector or use of any one of claims 11 to 21, wherein the other flavivirus is selected from the group consisting of west nile virus, yellow fever virus and japanese encephalitis virus.

23. A cell, preferably a human cell, comprising a poxvirus vector according to anyone of claims 1 to 8.

24. A kit for primary/booster immunization comprising in a first bottle/container the poxvirus vector of any one of claims 1 to 8 and 10 to 11 or the pharmaceutical composition of claim 9 for primary vaccination ("primary vaccination") and in a second bottle/container the poxvirus vector of any one of claims 1 to 8 and 10 to 11 or the pharmaceutical composition of claim 9 for secondary vaccination ("booster vaccination").

25. A poxvirus vector carrying DNA comprising an expression module comprising a transcriptional regulatory element and a shorter epitope chain encoding the complete flavivirus NS1 protein or which is still capable of eliciting an immune response; wherein the poxvirus from which the poxvirus vector is generated is derived from a virus of mvanbn V00083008 deposited at the european collection of cell cultures and has at least one of the following characteristics:

(i) reproductively replicating in Chicken Embryo Fibroblasts (CEF) and in baby hamster kidney cell line BHK, but not in human cell lines,

(ii) cannot replicate in mice with severely compromised immunity,

(iii) induces greater immunogenicity in a lethal challenge model compared to the known MVA 575 strain, and/or

(iv) At least substantially the same level of immunity is induced in the vaccinia virus prime/vaccinia virus boost regimen as compared to the DNA prime/vaccinia virus boost regimen.

26. Poxvirus vector according to claim 25, wherein the expression module further comprises a sequence encoding a further flavivirus peptide/protein, which comprises less than 40 amino acids, if derived from the E protein, but at least a glycosylation signal sequence.

27. The poxvirus vector of claim 26, wherein the human cell line is the human keratinocyte cell line HaCat, the human osteosarcoma cell line 143B and the human cervical adenocarcinoma cell line HeLa.

28. The poxvirus vector of claim 25 or 27, wherein the severely immunocompromised mouse is incapable of producing mature B and T cells.

29. The poxvirus vector of any one of claims 25 to 28, wherein the severely immunocompromised mouse is an AGR129 transgenic mouse.

30. The poxvirus vector of any one of claims 25 to 29, wherein the flavivirus is a mosquito-borne flavivirus.

31. The poxvirus vector of claim 30, wherein the mosquito-borne flavivirus is dengue virus.

32. The poxvirus vector of claim 31, wherein the dengue virus is dengue virus serotype 2.

33. Poxvirus vector according to anyone of claims 25 to 32, wherein the sequence encoding the flavivirus NS1 protein or a shorter epitope chain thereof still capable of eliciting an immune response is preceded by an ATG codon and a sequence encoding a glycosylation signal sequence, and wherein the coding sequence ends at a translation stop codon.

34. Poxvirus vector according to anyone of claims 25 to 33, wherein the transcriptional regulatory element is a poxvirus promoter.

35. Poxvirus vector according to anyone of claims 25 to 34, wherein the poxvirus vector is lyophilized.

36. A pharmaceutical composition comprising a poxvirus vector according to anyone of claims 25 to 35 and a pharmaceutically acceptable carrier, diluent and/or additive.

37. Poxvirus vector according to anyone of claims 25 to 35 as vaccine.

38. The poxvirus vector of claim 37, wherein the vaccine is used to vaccinate against the flavivirus or flavivirus serotype producing the nucleic acid or producing the NS1 protein or its shorter epitope chain that still elicits the immune response, and/or against one or more other flaviviruses and/or flavivirus serotypes.

39. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcriptional regulatory element and encoding an intact flavivirus NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response in the preparation of a vaccine against a flavivirus or flavivirus serotype that produces the NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, and/or against one or more other flaviviruses and/or flavivirus serotypes.

40. The use of claim 39, wherein the expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids, but at least a glycosylation signal sequence.

41. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcriptional regulatory element and encoding an intact flavivirus NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, in the manufacture of a medicament for the treatment or prevention of a flavivirus infection in an animal, including a human, in need thereof, wherein the flavivirus infection is an infection of a flavivirus or flavivirus serotype that produces the nucleic acid or produces the NS1 protein or a shorter epitope chain thereof that is still capable of eliciting an immune response, and/or an infection of another flavivirus and/or flavivirus serotype.

42. The use of claim 41, wherein said expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids, but at least a glycosylation signal sequence.

43. Use of a poxvirus vector carrying DNA comprising an expression component comprising a transcription regulatory element and a shorter epitope chain encoding the complete flavivirus NS1 protein or which is still capable of eliciting an immune response in the preparation of a medicament for the prevention of infection by multiple flaviviruses or flavivirus sera in an animal, including a human, in need thereof.

44. The use of claim 43, wherein said expression module further comprises a sequence encoding an additional flavivirus peptide/protein, which if derived from the E protein comprises less than 40 amino acids, but at least a glycosylation signal sequence.

45. Use according to any one of claims 39 to 44, wherein the poxvirus vector is a poxvirus vector according to one of claims 25 to 35.

46. The poxvirus vector of claim 38, the use of any one of claims 39 to 44, wherein the NS1 protein or its shorter epitope chain that still elicits an immune response is derived from one dengue virus serotype, and wherein the vaccine protects an individual against infection by at least two dengue virus serotypes.

47. The poxvirus vector or use of claim 46, wherein the vaccine protects the individual against infection by all serotypes of dengue virus.

48. The poxvirus vector or use of claim 46 or 47, wherein the dengue virus serotype and/or flavivirus from which the NS1 protein is produced is dengue virus serotype 2.

49. The poxvirus vector or use of any one of claims 38 to 47, wherein the other flavivirus is selected from the group consisting of West Nile virus, yellow fever virus and Japanese encephalitis virus.

50. A cell, preferably a human cell, comprising a poxvirus vector according to anyone of claims 25 to 35.

51. A kit for primary/booster immunization comprising in a first bottle/container the poxvirus vector of any one of claims 25 to 35 and 37 to 38 or the pharmaceutical composition of claim 36 for primary vaccination ("primary vaccination") and in a second bottle/container the poxvirus vector of any one of claims 25 to 35 and 37 to 38 or the pharmaceutical composition of claim 36 for secondary vaccination ("booster vaccination").