TWI336626B - Flavivirus ns1 subunit vaccine - Google Patents

Description

1336626 VI. OBJECTS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to NS1 proteins of flavivirus or portions thereof, particularly dengue viruses. The NS1 protein or portion of a flavivirus can be used for vaccination against the flavivirus and many other flaviviruses. The invention particularly relates to a NS1 protein of a dengue virus subtype or a portion thereof (particularly subtype 2) which can be used for vaccination against dengue viruses from all subtypes. More specifically, the present invention relates to a DNA comprising a marker of a yellow virus NS1 or a portion thereof, a vector comprising the DNA, and a vaccine comprising or expressing a flavivirus NS1. BACKGROUND OF THE INVENTION The pathogen of dengue is the dengue virus, which belongs to the Flaviviridae family (Burke and Monath, 2001). A particularly important subgroup of flaviviruses is called a flavivirus carried by mosquitoes, a mosquito-borne flavivirus. In addition to the dengue virus mentioned above, this group also contains other important viruses such as West Nile virus, sputum encephalitis virus and yellow fever virus (virus field' edited by FieldsB.N., Lippincott-Raven Press , 3rd edition 1996, ISBN: 0-7817-0253-4, pp. 931-1034). Typical diseases transmitted by these viruses are West Nile fever and West Nile encephalitis caused by West Nile virus, Japanese encephalitis virus, yellow fever caused by yellow fever virus, and dengue fever and dengue hemorrhagic fever caused by dengue virus ( DHF; see below) and dengue shock syndrome. An enveloped, single-stranded, positive-stranded RNA virus formed by three structural proteins: the coat protein (C), which forms a core 3 1336626 shell that binds to the viral genome, to which is attached a membrane (membrane) and The E (envelope) protein is surrounded by a fat bilayer. The genome is approximately llkb long and contains a single open reading frame encoding a polyprotein precursor of approximately 3400 amino acid residues. The individual viral proteins are produced by the action of cellular and viral proteases from the predecessor. The three structural proteins (c, Μ, and E) are derived from the N-terminal portion of the polyprotein and are followed by seven non-structural proteins: NS NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (Lindenbach and Rice, 2001). The glycoprotein NS1 present in all flaviviruses appears to be indispensable for virus viability. The dengue virus NS1 is secreted from infected mammalian cells in a soluble hexamer form (Flamand et al., 1999). This non-covalently bound hexamer complex is formed from 3 dimeric minor units and has a molecular weight of 310 kDa. If it is still the only viral intrinsic protein on the surface of the infected cell, dimerization is indispensable for the export of the NS1 protein to the cell membrane. In mammalian cells, non-insect cell lines can support dengue infection, and part of the delivered NS1 is released outside the cell. Extracellular NS1 is not secreted as a soluble protein (presented as a higher hexamer oligomeric form), but is secreted in association with microparticles rather than virions. In addition, NS1 has been found to circulate in the serum of patients infected with dengue virus, suggesting that secretion of NS1 may be an important factor in the infection of human hosts by flaviviruses. During infection with the flavivirus, the NS1 protein induces a strong antibody response to help clear the infectious virus in the host, possibly through the complement regulatory pathway (Schlesinger, JJ et al., 1987) and antibody-dependent cellular cytotoxicity (ADCC). (Schlesinger, JJ et al., 1993) ° 4 1336626 Dengue fever and its four serotypes, dengue virus serotype 1 ^en-n to dengue serotype 4 (Den-4), for the flavivirus infection Very importantly, the disease it produces ranges from symptoms like influenza to severe, or fatal, with dengue hemorrhagic fever in shock. The explosion of dengue fever is a tropical and subtropical zone with dense population and a large number of mosquito vectors. Important public health issues. /

The spread of dengue infection and other diseases caused by mosquito-borne flaviviruses has led to more efforts in the world to develop dengue fever vaccines for pre-denigative fever (DF) and dengue hemorrhagic fever (DHF). Protecting vaccinated individuals from vaccines that are caused by some or all of the mosquito-borne flaviviruses.

Although most DF cases show signs after first infecting any of the four serotypes, most DHF cases occur when the patient receives a second infection and the first infection with a different serotype of dengue virus. . This observation report yields the hypothesis that individuals infected with antibodies against a dengue serotype in a sequence of different viral serotypes at appropriate intervals may result in a specific number of cases. Antibody-dependent increase (fibrosis) has been obtained (4) outside of dengue fever and other enveloped viruses, and is considered an important mechanism in the development of disease. Alternatively, the idea is that 'DHF usually appears in geographically diverse areas with multiple (three or four) viral serotypes. In areas with localized rains such as in Southeast Asia, the incidence of specific years is higher in children, and many DHF cases are fewer in older groups. This is broadly consistent with the 'monthly increase in blood for dengue', which shows that (4) money can cause protective immunity. This is now different from that observed in viral infections such as A pear hepatitis virus. Clinical observations show that 'patients may suffer secondary DHF (Nimmannitya et al '1990)' however this is Very rare, and difficult to accurately identify the type of sera that causes the second and subsequent infections. So far, there have been no reports of four infections in the same individual, except that virtually all four dengue virus serotypes are transmitted in the same region. This implies that, in essence, two or three dengue virus serotype infections in the same individual may produce cross-reactive antibodies or even a reactive cytotoxic lymphatic response. It is possible to control bite protection against infection by the type of dengue virus sera that is essentially left. There are currently no approved dengue vaccines. Today, the prevention of dengue virus infection relies on the control of the main mosquito host, Egyptian black zebra. The lack of pesticide tolerance, technology and financial support will enable the local health sector to maintain an effective mosquito control program, and the continued geographical spread of both host and dengue viruses makes it particularly difficult to use current mosquito control programs. prevention. Therefore, the development of safe and effective vaccines against all four serotypes of dengue virus has been designated by WHO as the most cost-effective way to prevent dengue infection. The WHO recommends that the ideal vaccine against dengue and DHF should prevent infections caused by all serotypes, so that continuous infection does not occur. For each purpose, W0 98/13500 plans to use a recombinant modified vaccinia virus Ankara (MVA) that will express all antigens of the dengue subtype, or use four recombinant MVAs, each of which has at least one type of dengue fever Both of the k-prototypes of the virus sub-31 provide a very good vaccine against all dengue virus sub-5L. However, it is hoped to provide a single-unit vaccine that produces one against more than one yellow after application of 13 1336626. The virus or more than one type of dengue virus serotype, preferably an immune response against all dengue virus serotypes. C SUMMARY OF THE INVENTION 2 SUMMARY OF THE INVENTION Accordingly, the subject matter of the present invention provides a vaccine derived from a flavivirus or flavivirus subtype that is stable, easy to produce, and induces an immune response that protects the vaccinated individual It is not only free from the infection of the flavivirus or flavivirus subtype of the vaccine, but also from other flaviviruses or flaviviruses. A particular subject of the invention provides a vaccine derived from a flavivirus-borne flavivirus that protects a vaccinated individual from infection by a flavivirus or flavivirus subtype transmitted by a mosquito derived from the vaccine. And is free of infection by flavivirus or flavivirus subtypes transmitted by other mosquitoes. A further subject of the invention is to provide a vaccine derived from a subtype of dengue virus and to protect a body from infection by all dengue subtypes. I: Implementation:! DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS These objects have been solved by providing a virulence NS1 protein or a portion thereof and providing a DNA sequence each comprising a expression cassette encoding a flavivirus NS1 protein or a portion thereof. Specifically, for the purpose of providing a vaccine derived from a mosquito-borne flavivirus, it can be solved by providing a mosquito-transmitted flavivirus NS1 protein or a part thereof, which protects a body from being derived from the vaccine It is infected by mosquito-borne flavivirus and is also free of infection by other mosquito-borne flaviviruses, especially dengue virus, preferably dengue serotype 2 and individual relative DNA sequences. More specifically, for the purpose of providing a vaccine derived from a subtype of dengue virus, it can be solved by providing an NS1 protein or a part thereof of dengue virus (especially dengue serotype 2 and individual DNA sequences), The vaccine protects a body from at least infection with all dengue subtypes and is preferably free of infection by other flaviviruses (especially mosquito-borne flaviviruses such as sputum encephalitis virus, yellow fever virus and West Nile virus) ). As shown in more detail in the experimental section, the NS1 protein from the de novo manifestation of a dengue virus serotype is induced after vaccination with dengue virus serotypes 1, 2, 3 and 4 The NS1 protein plus NS1 from other members of the Flavivirus genus, such as sputum encephalitis virus, yellow fever virus and West Nile virus, produces a reactive antibody response. Thus, the NS1 protein from a dengue virus serotype is a universal DHF subunit for the simultaneous protection of all serotypes against dengue virus and further against other flaviviruses. Because there is no E protein involved in this unit vaccine strategy, there is no risk of antibody-dependent elevation (ADE) in any subsequent serotype of dengue virus, and therefore vaccine-associated DHF is transmitted in dengue fever. Natural outbreaks should not be induced during the period. According to a preferred embodiment, the invention relates to a DNA comprising a cassette which encodes at least one flavivirus NS1 protein or a portion thereof. The term "at least" as used herein means that the performance cassette further encodes an additional protein/peptide, which may be a separate protein/peptide or to the NS1 protein or portion thereof, as defined in detail below. As used herein, the term "DNA" refers to any type of DNA, such as single-stranded DNA, double-stranded DNA, linear or circular DNA or plastid DNA or a viral genome. Because the flavivirus is an RNA virus, the coding The DNA of the flavivirus NS 1 protein is a non-naturally occurring DNA such as cDNA or synthetic DNA. 1336626 4 The method of encoding the flavivirus NS1 protein or a portion thereof is described as "meaning position; I white or & part of the coding sequence Elements that control transcription, especially the initiation of transcription, are added before. An example of this transcriptional regulatory element is the priming of the prokaryotic promoter 7 . Compared to the slave nucleus (four) sub/(iv) subfamily human giant cell virus immediate early promoter/enhancer and as disclosed in the example section, such as? .5 starter, "four-toxic starter of the poisonous minimum starter. The phase of the minimum promoter of the plague virus is shown in Figure 2 and is the serial number: 9. If necessary, the wire can be cut into The elements of the termination, such as the pronuclear termination element or the eukaryotic multi-A signal sequence. The performance card E may only represent the yellow virus coffee white or part thereof or may be expressed together with - or a variety of additional flavivirus proteins / wins a protein or a portion thereof, wherein the β NS1 protein or a portion thereof and the additional protein/peptide are produced as an isolated protein 'successor or fusion protein/peptide form. If not defined herein, in the context of the present invention The term "consider" means at least iq amino acids, preferably 2 amino acids, more preferably a continuous amino acid sequence of 25 amino acids.

This additional flavivirus protein is not a complete E protein' because this protein seems to be involved in the development of Li. Thus, if the additional flavivirus peptide is derived from an E-protein, it should contain less than 4 () counts of less than 35 amino acids. If the long base of the amino-based warm sequence derived from the E-protein is expressed by deproteinization or its fraction, it should be confirmed that the amine-based long column does not contain an epitope involved in the production of fibrils and DHF. In addition to the NS1 protein or a portion thereof, if the performance card & also exhibits a flavivirus protein/_ in the form of an isolated protein/peptide, the performance of the sequence is in the sequence and coding of the encoded deproteinized portion or portion thereof. The additional 9-sequence of the flavivirus protein may contain - ribosome in place (IRES). The IRES element is known to the skilled person. Examples of IRES elements are the small RNA virus IRES element or the 5' uncoded region of the C! hepatitis virus. Optionally, a nuclear acid sequence encoding the NS1 protein or a portion thereof can be fused to a DNA array encoding an additional flavivirus protein/peptide, whereby the NS1 protein or a portion thereof is intervened with the additional A protein is formed between the flavivirus protein/peptide. The NS1 protein or a portion thereof is fused to the additional flavivirus protein/winning form of the fusion protein/peptide, and the respective coding sequences are fused in frame. In a preferred embodiment, the sequence encoding the glycated signal sequence of the E-protein is added prior to the DNA sequence encoding the NS1 protein or portion thereof. According to this specific example, a fusion protein is produced which comprises an E-protein saccharification signal sequence fused to the NS1 protein or a portion thereof. As indicated above, the £_protein-derived amino acid long column should be as short as possible and it should be excluded from the long-term listing of the amino acid to contain epitopes involved in ADE and j) HF production. The £_protein glycation signal sequence meets this requirement. Preferably, the performance cassette of the present invention comprises only the flavivirus sequence encoding the NS1 protein or a portion thereof. Thus, in a preferred embodiment, the performance cassette of the present invention does not exhibit any other peptide/protein ' from other phage viral portions that are NS2A or E proteins. In another optional embodiment, the DNA of the present invention exhibits an NS1 protein or a portion thereof which is in the form of a fusion protein having a protein/peptide which is not derived from flavivirus. The protein/peptide comprises a non-flavonoid signal sequence or a sequence for detecting or purifying a fusion protein such as a tag. 10 1336626 In a preferred embodiment of the invention, the general structure of the flavivirus sequence in the expression cassette is as follows: during native flavivirus infection, the virus produces a single polyprotein which is then first hosted by the host cell. The protease is interrupted and then cleaved by the virally encoded protease into the following proteins: C, PrM and Μ, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (protein sequence Dependent Protein Precursor Protein Arrangement). Therefore, the DNA sequence, particularly the cDNA sequence encoding the NS1 protein or a portion thereof, must be additionally appended with the "ATG" initiation codon. In a preferred embodiment, a saccharification signal sequence is added after the initiation of the ATG, whereby the newly synthesized NS1 protein is saccharified in the endoplasmic reticulum. This sequence of signals is known to those skilled in the art. Finally, the cassette encoding the protein requires a stop codon, which can be a TAG added to the 3' terminus of the protein encoding the cDNA sequence. For example, the "ATG+signal sequence" element used in the present invention is derived from the hydrophobic C-terminus of the E protein (the last 28 amino acids, which are dengue virus neonatal scorpion breeds (,, NGC cultivar, "gene The database accession number AF038403) is based on amino acid M (ATG). A typical performance card of the present invention is shown in Figure 2 and is represented by SEQ ID NO: 9 and SEQ ID NO: 10. Thus, in short, this A specific example relates to a DNA comprising a cassette which comprises a sequence encoding a flavivirus NS1 protein or a portion thereof, wherein the coding sequence is preceded by a start codon ("ATG") and a coding sequence. The sequence of the signal sequence for saccharification is preferably derived from an E-protein as defined above and wherein the coding sequence is terminated by a translational stop codon (1A, 1C and 2, SEQ ID NO: 5-10). The DNA sequence of the invention encodes a flavivirus NS1 virus or a portion thereof. The term "flavivirus" means any flavivirus. More preferably, the term "flavor virus" means, for example, 11 West Nile virus, Japanese encephalitis virus, yellow fever. Virus and boarding A mosquito-borne virus-borne virus that is derived from a mosquito-borne deproteinized portion or a portion thereof that is encoded by the DNA of the present invention, and which protects the vaccinated individual from the virus or virus that generates the vaccine in the street. Subtype infection, and exempt from the infection of the vaccine... a mosquito-borne virus or other viral subtype infection. The NS1 protein may preferably be any dengue virus subtype. More preferably, g NS1 The protein coding sequence is derived from a species such as the dengue virus neonatal squirrel breed (,, NGC cultivar ", Genodata Accessory No. 38_ of the dengue subtype 2. The term "subtype," and "serum type" in this invented book It can be used interchangeably. The term "NS1 protein or a part thereof", "part of it" means a long column of amino acids of the protein, which is long enough to induce a specific immune response against the derivation of the "part" thereof. NS1 protein. If the flavivirus is a dengue virus, the long-term amino acid should be immune to NS1 protein in all vaccinated animals (including human (5) material line against all dengue-detoxification subtypes). reaction Amino acid sorghum shows in the Examples section how a person skilled in the art can vaccinate the NS1 protein or a portion thereof to induce an immune response to all of the Denga subtypes. A preferred embodiment, the huanghu sequence encodes the entire NS1. Briefly, in a preferred embodiment of the invention, the phylum comprises a sputum containing a mosquito-borne flavivirus NS1 or a portion thereof, wherein the flavivirus is preferably dengue fever, more preferably dengue fever. Viral subtype 2, and wherein the expression of the NS1 protein or a portion thereof is controlled by eukaryotic transcriptional regulatory elements. More preferably, the DNA of the present invention is NS1 protein in the form of a fusion protein having a saccharide signal sequence. Or part thereof.

The present invention further refers to a carrier comprising the DNA of the present invention, and the term "13 1336626" means "known to those skilled in the art. The carrier may be a carrier of a plastid such as 邠R322 or a pUC series. Preferably, the vector is a viral vector. In the context of the present invention, the term 'viral vector' or 'viral vector' means an infectious virus comprising a viral genome. In this case, the DNA of the present invention is It is to be selected into the viral genome of the respective viral vector. The recombinant viral genome is then packaged, so that the obtained recombinant vector can be used to infect cells and cell lines, particularly living animals infected with humans. A typical viral vector according to the present invention is an adenovirus vector, an enterovirus vector or a vector based on adeno-associated virus 2 (AAV2). Preferably, it is a carrier of poxvirus. The poxvirus should preferably be gold wire. It is better to modify the vaccinia virus Ankara (UVA) (Sutter, G et al., [1994], Vaccine 12·· 1032-40). Typical MVA varieties It is MVA 575, which has been deposited at the European Animal Cell Culture Collection Center under the registration number ECACC V00120707. More preferably MVA-BN or its derivatives, which was described on November 22, 2001 by the applicant Bade The Belgian Nordic Research Institute GmbH, titled "Modified Bovine Ankara Virus Variant", is filed in the PCT application filed by the European Patent Office. MVA-BN has been deposited in European animals. In the cell culture collection center, the registration number is ECACC V00083008. By using MVA-BN or its derivatives, additional technical problems have been solved, and a special safe virus vaccine against the flavivirus is provided, as MVA- has been shown. The BN viral vector is a completely attenuated virus derived from the modified vaccinia Ankara virus and characterized by the loss of its ability to replicate efficiently in human cell lines. MVA-BN is less than any due to its lack of replication in humans. Other known vaccinia virus species are also safe. In a preferred embodiment, the present invention relates to MVA-BN and MVA-ΒΝ derivatives containing DNA according to the invention. Biological carrier. Characteristics of MVA-BN, a description of the bioanalysis that allows evaluation of whether MVA is a derivative of MVA-BN or MVA-BN, and a method allowing obtaining MVA-oxime or a derivative thereof are shown in the following description section and examples. "M" derivative of the term "virus", such as the "derivative" of the virus of the registration number ECACC V00083008 (ie, a derivative of MVA-ΒΝ), means to display at least one characteristic of the registered variety, but in One or more of its genomes show differential vaccinia virus. Preferably, the derivative has at least two, more preferably derivatives having at least three, more preferably all of the following four MVA-oximes. In particular, the derivative of MVA-BN has substantially the same replication characteristics as MVA-ΒΝ. The virus having the same "replication characteristics" as the registered virus means that in the CEF cells and cell lines BHK, HeLa, HaCat and 143B, there is a virus having a similar amplification ratio to the registered variety, and when the AGR129 gene is transferred When the rat model is detected, it shows similar replication in vivo. The characteristics of MVA-ΒΝ are as follows: - can be effectively replicated in chicken embryonic fibroblasts (CEF) and baby hamster kidney cell line BHK (ECACC 85011433), but No replication capacity in the human cell line HaCat (Boukamp et al., 1988, J Cell Biol 106(3): 761-71), - unable to replicate in vivo, - compared to the conventional MVA575 (ECACC V00120707), Induction of higher immunogenicity in a mutual stimulation model and/or induction of at least substantially the same level of immunity in vaccinia virus primary/vaccinia virus push-up therapy compared to DNA-primary/vaccinia virus push-up therapy The term "capability without effective replication" means a virus of the invention exhibiting a replication rate of less than 1 in the human cell line HaCat (Boukamp et al., 1988, J Cell 14 1336626).

Biol. 106(3): 761-71. Preferably, the virus used as the vector of the present invention has an amplification ratio of 0.8 or less in the human cell strain HaCat. The "expansion ratio" of a virus is the ratio of the number of viruses (inputs) generated from an infected cell (output) relative to the number (input) originally used to infect the cell at the first position ("amplification ratio"). A ratio of "1" between the output and the input is defined as an amplification state in which the number of diseased Qin derived from the infected cells is the same as the number originally used to infect the cells. This state implies the fact that infected cells allow viral infection and viral replication. The best MVA vectors, particularly MVA-BN and its derivatives, do not replicate efficiently in HaCat cells. In particular, MVA-BN showed an amplification ratio of 〇.〇5 to 0.2 in human embryonic kidney cell line 293 (ECACC No. 85120602). 0至范围内。 The human osteosarcoma cell line 143B (ECACC No. 91 112502), the ratio is in the range of 0. 0 to 0.6. As for human cervical adenoma cell line HeLa (ATCC No. CCL-2) and human keratinocyte cell line HaCat (Boukamp et al., 1988, J Cell Biol. 106(3): 761-71), amplification thereof The ratio is in the range of 0.04 to 0.8 and 0. 02 to 0.8. The amplification ratio of MVA-BN in African green monkey kidney cells (CV1: ATCC No. CCL-70) was 〇.〇1 to 0.06. Therefore, 'MVA-BN does not replicate efficiently in any of the tested human cell lines. The amplification ratio of MVA-BN in chicken embryonic fibroblasts (CEF··primary culture) or juvenile gas kidney cell line BHK (ATCC No. CRL-1632) was clearly greater than 1. As outlined above, a ratio greater than "1" means efficient replication because the number of viruses produced from infected cells is increased compared to the number of viruses used to infect cells. Therefore, the virus can be easily propagated and expanded in BHK cells at a ratio of greater than 500 in the primary culture of cef' and at a ratio greater than 50. 15 1336626 In the context of defining MVA-ΒΝ, the term “unable to replicate in vivo” means that the virus cannot replicate in human and mouse models as described below. The "unable to replicate in vivo" is preferably determined in mice that are unable to produce mature B and T cells. An example of such a mouse is the genetically transgenic mouse model AGR129 (available from Mark Sutter, University of Zurich, Viral Research Institute). This mouse cultivar has a genotypic interference with the gene in the ifN receptor type I (IFN-α/β) and type II (IFN-γ) genes and RAG. Due to these glucosides, the mouse lacks the IFN system and is unable to produce mature B and T cells and is itself a serious immune abandonment and highly susceptible to replicating virus. In addition to AGR129 mice, any other mouse species that are unable to produce B and T cells and are severely immune to their own and highly susceptible to replicating viruses can be used. In particular, the virus according to the present invention does not poison the mouse for at least 45 days, preferably 60 days, preferably for a period of 90 days after infecting AGR129 mice by intraperitoneal injection of 1 〇 7 pfu of virus. . Preferably, the virus exhibiting "unable to replicate in vivo" is further characterized as '45 days after infection of AGR129 mice by intraperitoneal injection of 1〇7 pfu of virus', preferably 60 days, preferably At 90 days, no virus recovered in the organs or tissues of the mouse. MVA-ΒΝ and its derivatives are preferably characterized by a higher immunogenicity than the known MVA 575 varieties, as measured by a lethal-stimulated mouse model. In this model, mice that were not vaccinated died after being infected with a vaccinia variety that was fully replicated, such as the Western Reserve variety L929 TK+ or IHD-J. The fully vaccinated vaccinia virus infection is called "stimulation" in the description of the lethal stimulation model. After four days of stimulation, the mice are usually poisoned and used for standard speckle analysis by using VERO cells. The virulence titer of the disease 16 1336626 can be measured in the ovary. The virus titer in the non-vaccinated mouse and the mouse vaccinated with the poxvirus of the present invention is determined. More specifically, the virus according to the present invention is characterized by In this test, after inoculation with 102TCID5»/ml of the virus of the present invention, the virus titer in the ovary is reduced by at least 70%, preferably by at least 80%, more preferably by at least 90% compared to the non-vaccinated mouse. MVA-BN or a derivative thereof is preferably characterized in that it induces at least substantially the same immunity in vaccinia virus primary/bovine virus boost therapy as compared to DNA-primary/vaccinia virus push-up therapy. In comparison with the DNA-primary/vaccinia virus push-up therapy, if the vaccinia virus primary/vaccinia virus push-up therapy is used, the following two methods are used (, analysis method 1 ", and, analysis method 2") Any one, preferably In parallel, when the CTL reaction measured in the system is at least substantially the same, then a vaccinia virus is considered to be compared to the DNA_primary/bovine: viral boost therapy, and the bovine viral first time/vaccinia Viral push therapy induces at least substantially the same level of immunity. More preferably, at least one assay is performed after the vaccinia virus primary/vaccinia virus is boosted compared to the DNA-primary/vaccinia virus push-up therapy. The CTL response was higher. Best, the CTL response was higher in the following two assays. Analytical method 1: For vaccinia virus primary/vaccinia virus push-up administration, by intra-abdominal injection 107 TCIDsd, as in the present invention, will display a murine polyhedrin vaccinia virus (described in Thomson et al., 1988, J. Immunol. 160, 1717) for the first time - immunization of 8 week old BALB/c (H-2d) mice, and After three weeks, the same way, the same number of viruses were used to boost-immunize the mouse. For this purpose, it is necessary to construct a recombinant vaccinia virus system that expresses the polyhedron. Known by the skilled person, and As described in detail below, when the DNA primary/vaccinia virus push-up therapy is performed, the initial inoculation is performed by intramuscular injection of the mouse, and the 50 mg of the mouse exhibits the same antigen as the vaccinia virus; / Vaccinia virus pushes up the same method to carry out the promotion of vaccinia virus. In the disclosure of Th〇ms〇n et al. cited above, the plastid of the polyhedron is also explained. The development of the CTL response to the anti-antigenic epitopes SYIPSAEKI, RPQASGVYM and/or YPHFMPTNL in both therapies was measured two weeks after the boosted administration. Preferably, the determination of the CTL reaction is performed using Eusp〇T (described in

Schneider et al., 1998, Nat. Med. 4, 397-402, and a specific virus of the present invention are outlined in the Examples section below. The characteristics of the virus according to the present invention, in this experiment, are evaluated by the vaccinia virus primary/vaccinia virus boosting administration when evaluated by the number of cells producing ΙΡΝ_γ/1 〇 6 spleen cells, against the above mentioned The CTL immune response of the epitope is substantially the same as that caused by the initial administration of the DNA/vaccinia virus (see also the experimental part). Analytical Method 2: This analysis basically corresponds to Analytical Method 1. However, instead of intravenously administering 1〇7 TCIDm vaccinia virus as in Analytical Method 1, a 〇8 TCIDsd, such as the bovine mark virus of the present invention, is administered subcutaneously for primary immunization and boosting immunization. The characteristics of the virus according to the present invention, in this experiment, when evaluated by the number of cells producing IFN-γ Λ〇6 spleen cells, against the above mentioned bovine cancer virus primary/bovine cancer virus boost The CTL immune response to the epitope that is caused is substantially the same as that caused by the promotion of the dna primary/vaccinia virus (see also the experimental part). It is well known to those skilled in the art how to obtain the properties of MVA-BN and its derivatives as indicated above: The method for obtaining this virus may comprise the following steps: 18 1336626 - Introduction of a known vaccinia virus variety' Preferably, MVA 574 or MVA 575 (ECACC V00120707) is in a non-human cell in which the virus is capable of efficient replication, wherein the non-human cell is preferably selected from CEF cells and cell line BHK, - isolated/rich from such The virions of the cells, and - whether the virus obtained by the assay has at least one biological property as desired above, wherein the steps described above can optionally be repeated until a virus having the desired replication characteristics is obtained. Methods for inserting a DNA of the present invention into poxvirus DNA and methods for administering a recombinant cancer virus are well known to those skilled in the art. In a recombinant vaccinia virus, the performance of the DNA of the present invention is preferably under the transcriptional control of a poxvirus promoter, preferably a vaccinia virus promoter, but is not absolute. Preferably, the DNA of the present invention is 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 a position in the MVA genome where deletion occurs naturally (disclosed in pCT/Ep96/〇2926). Briefly, a preferred embodiment of the invention provides a vector comprising a DNA according to the invention, wherein the vector is MVA BN or a derivative thereof and wherein the DNA according to the invention comprises - encoding a dengue virus, in particular a dengue virus The performance of serotype 2 NS1 protein or a portion thereof is defective. In a preferred embodiment, the invention relates to an NS1 protein or a portion thereof encoded by a DNA according to the invention or a vector according to the invention. For the definition of the eye or a part thereof of the present invention, reference is made to the above section of the specification, wherein the D N A of the present invention has been defined by the product represented by the DNA. Accordingly, the following brief description of the white matter of the egg 19 1336626 according to the present invention is not to be considered as a limitation of the present invention. Briefly, the protein of the present invention may be an isolated NS1 protein or a portion thereof encoded by any flavivirus. Preferably, the NS1 protein or portion thereof is derived from a dengue virus, and more preferably from the dengue virus subtype 2. The protein of the invention may comprise only the amino acid sequence of a viral NS1 protein. In a preferred embodiment, the nsi protein may comprise additional amino acids which are necessary for efficient expression of the protein. Examples of the amino acid/amino acid sequence are shown above and include a methionine at the N-terminus of the protein from the tarcode of the ATG codon to which it is added, and a C- from the E-protein. The terminal-derived amino acid sequence functions as a signal sequence for saccharifying the protein of the present invention, and other signal sequences are also included in the scope of the present invention. In an optional embodiment, the NS1 amino acid sequence or a portion thereof can be fused to other proteins/peptides. Examples of fused partners are sequences that allow for the identification of proteins, such as tags or other flavivirus proteins or portions thereof. In a preferred embodiment, the invention relates to a DNA, vector or NS1 protein or a portion thereof of the invention as a vaccine. A "vaccine" is a compound that is a DNA, protein, vector or virus that induces a specific response. According to an optional embodiment, the "vaccine" of the present invention is based on the dengue virus NS1 protein or a portion thereof which induces an immune response against all n-type proteins of dengue virus subtypes. In particular, it has been shown that the NS1 protein of the dengue virus subtype, the specific subtype 2, induces an immune response against the NS1 protein of all dengue virus subtypes, preferably against other mosquito-transmitted viruses. . As described above, the inventors of the present invention have found that the N-magic 20 1336626 protein of the flavivirus of the present invention or a portion thereof induces an immune reaction against the NS1 protein of other flaviviruses. As the above, "the flavivirus, preferably a mosquito-borne flavivirus. In other words, the inventors of the present invention have found that in the present case - the optional heterogeneous example is the present invention.

The NS1 protein or part of the mosquito-borne flavivirus, which embres to produce a mosquito against the mosquito-borne flavivirus derived from the vaccine, and the anti-aliasing against other transgenic mosquito-like flaviviruses. A vaccine derived from a relatively spreader of flavivirus may be used as a counter- or a plurality of mosquito-borne vaccines. The term "a vector derived from a flavivirus" or a similar term herein refers to a vector comprising the purine of the present invention as indicated above (eg ELISA virus vector or plastid. Thus, the term means a vector inlay rather than a vector backbone. An example of a "vector derived from a flavivirus" is a prion vector such as MVA, which contains - the expression n represents a card containing 4 a viral promoter, a sequence encoding a flavivirus NS1 protein or a fragment thereof, wherein the sequence encoding the deproteinized portion of the flavivirus or a portion thereof is preceded by a sequence of a Norma and a squaring saccharification signal sequence and a sequence thereof The terminal of the code sequence is a translation stop codon.

Therefore, the single-subunit disease with the 'vector' or the deproteinized protein that can counteract the broad yellow-skinned subtypes of the yellow scales encodes the de-protein or part of the flavivirus or flavivirus subtype. Zhao, or 疋 from the yellow virus or subtype of milk ι * white or as a vaccine against other Huangxian and yellow disease "like dengue virus subtype 2 can be used as anti-subtype 3 and ^ and anti-subtype 2 Vaccine. It is more usable. Ding Jifei is used to protect one body against other flaviviruses such as western poison. Wei's disease In a preferred embodiment, the DNA of the present invention is made into a vaccine. 1336626 The exposed viscera of the eukaryotic expression card E of the present invention, particularly intramuscular injection of DNA, can result in the performance of the protein encoded by the performance card, as is known to those skilled in the art. The immune system, and produces a specific immune response. In the specific example, the vaccine is vaccinated by administering the vector of the present invention 'special S virus vector', preferably a viral vector, preferably a nuclear virus. The carrier is like MVA The virus of the present invention is converted into a physiologically acceptable form in the preparation of a vaccine based on vaccinia virus. This preparation is based on the experience in preparing a poxvirus vaccine against smallpox. In Stickl, H. enriching, [1974] Dtsch· med. Wschr. 99, 2386-2392). For example, the purified virus is stored at -80 ° C ' at a potency of 5 x 108 TCID 5 〇 / ml at about 1 〇 mM di-methylamino oxime (Tris), 140 mM NaCl pH 7.4. In the preparation of vaccines, such as 1 〇 2 - 108 virions in 1 〇〇 ml, there are 2% protein glands and 1 % human albumin phosphate buffered saline (PBS) is lyophilized in ampoules, preferably glass ampoules. Optionally, the vaccine can be produced by gradual freeze-drying of the virus in the formulation. Additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gases, stabilizers or proteins suitable for in vivo administration (eg human serum proteins) After the glass The ampoule is sealed and can be stored at 4 ° C and room temperature for several months. However, as long as it is not necessary, An Yin is better stored at a temperature below -20. (: at the time of inoculation, it will be The lyophilizate is dissolved in a liquid solution of 0.1 to 0.5 ml, preferably a physiological saline solution or a buffer, for systemic or topical administration, that is, by non-oral, intramuscular or other practitioners known as 22 1336626 Administration of administration mode. The number of administration modes, dosages and administrations can be optimized by those skilled in the art. The optimal administration of poxvirus vectors is subcutaneous or intramuscular administration. A specific embodiment of the present invention relates to a vaccine kit comprising the MVA-DN viral vector of the present invention for use in a first vial/container The first vaccination of the first vaccination in the first ") and the second vaccination in the second small glass bottle / container (,, push "". • If the vaccine is an MVA-BN vector comprising the DNA of the present invention or a purine thereof, a specific specific example of the present invention relates to the first vaccination ("primary vaccination") and the second vaccination (" Pushing a Vaccination") administering a therapeutically effective amount of the vaccine. Thus, in a particular embodiment of the vaccine, the invention relates to a vaccine comprising a DMA, vector or NS1 protein of the invention or a portion thereof, and the DNA, vaccine or protein is used The use of a vaccine. According to a preferred embodiment, the invention relates to the use of the DNA, vaccine or protein for the preparation of a vaccine, wherein the NS1 protein or a portion thereof, the NS1 protein encoded by the DNA or the vector, or a portion thereof, is derived from a dengue. Type, and wherein the DNA, the vector or the NS1 protein or a portion thereof is used as a vaccine against all dengue virus subtypes. The best dengue virus subtype is subtype 2. The present invention relates to a method for treating or preventing a flavivirus infection, which comprises vaccinating an animal, such as the above-mentioned vector or the NS1 protein as described above, in an animal including a human in need of treatment or prevention of flavivirus infection. Or part thereof. In particular, the invention relates to a method as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a portion thereof is derived from a 23 1336626 dengue virus subtype, and wherein the DNA, The vector or the NS1 protein or a portion thereof is used as a vaccine against all dengue subtypes. BRIEF DESCRIPTION OF THE INVENTION A DNA comprising a performance cassette encoding at least one flavivirus NS1 protein or a portion thereof. A DNA as described above, wherein the expression cassette comprises a transcriptional regulatory element of eukaryotes and a sequence encoding a flavivirus NS1 protein or a portion thereof. A DNA according to the above, wherein the sequence encoding the NS1 protein of the flavivirus or a portion thereof is preceded by an ATG codon and a sequence encoding a glycation signal sequence, and wherein the coding sequence ends with a translated stop codon. A DNA as described above, wherein the transcription regulating element of the eukaryotic organism is a trace virus promoter. A DNA as described above, wherein the flavivirus is a mosquito-borne flavivirus. A DNA as described above, wherein the flavivirus is a dengue virus. DNA as described above, wherein the dengue virus is a dengue virus subtype 2. A vector comprising the DNA as described above. A vector as described above, wherein the vector is a poxvirus vector. A vector as described above, wherein the poxvirus is a vaccinia virus, in particular a modified vaccinia virus Ancara (MVA). MVA-BN or its derivatives, especially MVA 575 (ECACC V00120707), preferably MVA-BN (V00083008). An NS1 protein or a portion thereof, which is encoded by DNA as described above or as described above. The DNA as described above, such as the above vector or the NS1 protein line as described above, is used as the vaccine. A DNA, vector or NS1 protein as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a portion thereof is derived from a mosquito-borne flavivirus, wherein the DNA, the vector, the DNA The NS1 protein or a portion thereof is used as a vaccine against a mosquito-borne flavivirus derived from a vaccine (i.e., the DNA, the vector or the NS1 or a portion thereof) or against other mosquito-borne flaviviruses. A DNA, vector or NS1 protein as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a portion thereof is derived from a dengue virus subtype, wherein the DNA, the vector, the NS1 protein Or a portion thereof is used as a vaccine against all dengue virus subtypes. The dengue virus-based dengue virus subtype 2 is preferably derived from the vaccine. A vaccine comprising DNA as described above, such as the vector described above, such as the NS1 protein described above. A DNA such as the above, such as the above-described vector, such as the above-described NS1 protein, is used for the preparation of a vaccine. The use as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a portion thereof is derived from a dengue virus subtype, wherein the DMA, the vector, the NS1 protein or a portion thereof is used As a vaccine against all dengue virus subtypes. The use as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a portion thereof is derived from a mosquito-borne flavivirus, wherein the DNA, the vector or the NS1 protein or a portion thereof It is used as a flavivirus 25-transmitted flavivirus against a derivative vaccine (ie, the DNA, the vector or the NS1 or a portion thereof) and a vaccine against other mosquito-borne flaviviruses. A method for treating or preventing a flavivirus infection, comprising vaccinating a DNA as described above, such as the above vector, the NS1 protein as described above or a part thereof to an animal in need of treatment or prevention of the flavivirus infection, including human . A method according to the above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the DNA or the vector or a part thereof is derived from a dengue virus subtype, wherein 4 DNA, s-negative vector,; Partially used as a vaccine against all dengue virus subtypes. The use as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the sputum or the vector or a part thereof is derived from a mosquito-borne flavivirus, wherein the DNA, the vector or the prion protein or It is used in part as a mosquito-borne flavivirus against a derivative vaccine (ie, the DNA, the vector or the NS1 or a portion thereof) and a vaccine against other mosquito-borne flaviviruses. EXAMPLES The following examples will further illustrate the invention. They are fully understood by those familiar with the art. The embodiments provided are not to be construed as limiting the implementation of the techniques provided herein to such embodiments. Example 1: Composition of mBN07 Detailed description of L NS1 antigen (Fig. 1) This example relates to NSI of serotype 2 from the Scorpio mouse c variety _NGC variety (e.g., gene library sequence AF038403). Since the nsi protein of the flavivirus is produced as part of a polyprotein precursor, the NS1 gene is not preceded by the "ATG" start codon in terms of the corresponding DNA. Therefore, the 'CDNA sequence encoding the NS1 protein must be added "ATG" Start 26 1336626 codon. This is followed by a signal sequence whereby the newly synthesized Ns! protein is glycosylated in the endoplasmic reticulum. Finally, the protein-encoding cassette requires a stop codon. And in this embodiment, the TAG is added to the 3' terminus of the protein-encoding cDNA sequence. The "ATG+ signal sequence" element used in the embodiment of the present invention is derived from the hydrophobic c-terminus of the E protein ( At least 28 amino acids, starting with amino acid M (ATG) for NGC varieties. Figure 1A shows the exact signal sequence + NS1 sequence as an embodiment of the invention (see also sequence number 5 and 6) The "signal sequence + NS1" nucleotide coding sequence is obtained by RT-PCR amplification of the dengue NGC RNA genome by using the following primers: D2NSb1 above: 5'-ACA^^rCTGAATGAATTCACGTAGCACCTCA- 3, (sequence number 4) oblique Word part: Bgl II restriction endonuclease recognition position. Underlined is the start codon. D2NS Bu 2 below: 5' -AAT^^7C7CTACTAGGCTGTGACCAAGGAGTT-3, (SEQ ID NO: 3) Italic subsection: Bgl II The restriction enzyme recognizes the position. The underlined is the stop codon. According to the manufacturer's recommended instrument, the RT-PCR amplification can be used from

Roche Molecular Biochemical's Titan One Tube RT-PCR kit (catalog number 1-939-823) was performed. However, essentially any commercial or uncommercial RT-PCR kit can be used instead. 27 1336626 Thereafter, the RT-PCR product can be colonized into the BamHI position that occurs in any of the majority of the commercially available bacterial selection plastids. However, in this example, it is colonized. Enter PAF7 'to select for PaF7D2NS1 - see Figures 1B and 1C for the detailed sequence of PAF7D2NS1. The ID plot shows the NS1 amino acid sequence and the hydrophobic ink dot containing NS1 plus the signal sequence from the C-terminal amino acid coding sequence of the E protein. The short F-terminal hydrophobic region refers to the signal sequence. 2. Details of the NS1 performance cassette (Fig. 2) is the "signal sequence + NS1" from the poxvirus vector such as canarypox, poultry pox, bovine colonization or MVA, at the 5' end of its cDNA A poxvirus promoter is required. A multi-adenosed signal sequence is not necessary when all of the poxvirus-synthesized RNAs are viral-encoded enzymes that do not require a polyA extra sequence to perform this function. Any cancer/virus promoter can be used to express this card. Fig. 2 and SEQ ID NOs: 9 and 10 show the nucleotide sequences of "poxvirus promoter + signal sequence + NS1" cassette as an example of the present invention. With respect to the examples used in the present invention, the "signal sequence + NS Γ is further amplified from the NS1 plastid by PCR using primers OBN338 and OBN345. The oBN345 primer contains the nucleotide sequence of the minimal promoter element 5' of the colony virus to the target sequence within the plastid. The sequence of the standard for the binding of the OBN345 primer is approximately 40 nucleotides upstream of the start codon of the signal sequence. This is used to determine that transcription of RNA contains a long list of non-protein coding sequences prior to the ATG start codon of the signal sequence. PCR primer with Ps promoter: oBN338: 5'-TTGTTAGCAGCCGGATCGTAGACTTAATTA (30) (sequence 28 1336626 No. 1) oBN345: 5, -CAAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAmA ACACGATAATACCATGG -3' (sequence number 2) Underlined nucleotides represent the pox The smallest promoter sequence of the virus, j. The binding temperature for the first five cycles of the PCR amplification reaction was calculated by binding the OBN345 to the nucleotide sequence of the same sequence in the selection vector. 3. Integrate the NS1 performance cassette into MVA (Fig. 3). The PCR amplified product is selected as the terminal Xho I position by the flat blunt end into the flat incision of the plastid ρΒΝχ〇7 (see Figure 3a) to form the plastid Body pBN41 (see figure 3a). pBN41 was used to integrate the "promoter + signal sequence + NS1 " cassette into the deletion position 2 of MVA by homologous recombination. The basic characteristics of pBN41 (see Fig. 3a) are as follows: - The plastid skeleton is from the stratagene of this town ice SK-plus (Genbank VB0078). -D2F1: deletion of 2 flanking 1 homologous recombination arms. This represents the nuclear: acid sequence from 20117 to 207 of the gene library sequence U94848. -PPr: Pox virus promoter. -NPTII··New Huisu filled with enzyme-encoding protein-making sequence (protein coding sequence of gene library v〇〇618). -IRES: ribosome in vivo sequence from encephalomyocarditis virus (Jang et al., 1989, Gene Database M16802). -EGFP: Elevated green light protein protein-drinking sequence (protein library sequence U57609 protein coding sequence - nucleotide 675 to nuclear per acid 1394). 29 1336626 -NSl: The "signal sequence + NS1" protein coding sequence from the dengue NGC variety. -D2F2: deletion of 2 flanking 2 homologous recombination arms. This represents the nucleotide sequence of 20719 to 21343 from the MVA gene library sequence 1) 94848.

AmpR: pBluescript is an aminopyrazine resistance gene. 3. 1 by the homologous recombination of dengue & acne promoter + signal sequence + NS1 " insertion of MVA deletion position 3.1.1 by homologous recombination to the MVA genome The above integration vector pBN41 is used for the side of pBN41 Homologous recombination between the 1 and the lateral 2 arms and the same target sequence in the MVA genome, the dengue NS1 expression cassette and the reporter cassette (pox promoter + NPT II-IRES-EGFP) were integrated into the MVA genome . This was achieved by transfecting the linear integration vector into chicken embryonic fibroblasts (CEF) that had previously been infected with a low amount of repeated MVA (MOI, e.g., 0.01 infectious units per cell). After 48 hours of infection or when the infection had reached confluence, the virus extract was prepared and stored at -20 °C for selection and purification of the desired recombinant MVA (rMVA). 3.1.2 Selection of rMVA and purification of selected strains Exclusion of non-recombinant MVA (empty vector virus) and amplification of rMVA by the presence of G418 (the number of G418 must be optimized until it is decided not to kill The highest dose of CEF cells is achieved by infecting fusion chicken embryonic fibroblast (CEF) cells with low Μ. Any virus that does not contain the integrated Νρτ n gene will not replicate in the presence of G418 added to the cell maintenance medium, and 418 will inhibit DNA replication, but since the CEF cells will be immobile, non-replicating, Its etc. will be 13 1336626 not affected by G418 again. Due to the enhanced expression of the fluorescent green protein, CEF cells infected with rMVAs can be observed under a fluorescence microscope. The virus extract from this homologous recombination step must be sequentially diluted and used to infect untreated CEF cells in the presence of G418 and cover the gelatin gum at a low melting point for m days. The vegetable gelatin board was placed under fluorescent .4 micromirrors to observe the single-green clear image of the infected cells. These were labeled and the gelatin plug containing the cells infected with clear imaging was removed and placed in a 1.5 ml microcentrifuge tube containing sterile cell maintenance medium. The virus is released from the gelatin plug by 2-3〇13: freeze-thawing the tube 3. The best selection strain or the selection strain is further pCR under the vegetable gum Knockout to purify and purify until there is no evidence of contamination of the vector (3 to 3 rounds of colonization and purification). The plants are then expanded for further stringent testing to correct the inserted configuration, foreign Sequence identification of the promoter gene cassette and analysis of the performance of RT-PCR. After these analyses, only one selected strain will be further amplified under the selection of G418, and a major stock solution will be prepared for further preparation. Characteristics and immunogenicity studies. The recombinant MVA having the dengue expression cassette inserted in the present invention is referred to as mBN07. Figure 3b shows the configuration of the foreign sequence inserted in mBN〇7. The expression of the true NS1 NS1 protein from this recombinant MVA (mBN07) was confirmed by standard Western blotting and was confirmed without denaturation. Figure 4 shows that purified mBN07 was used to infect mammals. Tissue culture cells (eg BHK-21 cells, MOI The result of NS1 expression after 1.0 infection unit per cell. The protein extract of Rough 31 1336626 was prepared from the infected cells after 24-3 G hours of money, at which time some extracts were containing 2 巯 groups. Ethanol (2_ME) or SDS-PAGE gel loading buffer without 2-ME is mixed. These samples, which are positive control groups, are supplemented with protein extracts from cells infected with dengue NGC varieties (mosquito cell lines). The SDS-PAGE gel was separated by electrophoresis and then blotted onto a nitrocellulose membrane. The membrane was probed with a monoclonal antibody against dengue NS1. Figure 4 shows that the NS1 line expressed by mBN07 is resistant. - Dengue NS1 monoclonal antibody to identify and form the correct dimeric pattern similar to NS1 from dengue-infected cells (compared to no boiled '2B-free mBN〇7 lane with no boiled, not 2-ME DEN2 Lane). Figure 4 shows that the dimeric form is separated into a monomeric form in the case of denaturation (see the boiled mBN07 lane containing 2_贶). The NS1 expressed in cells infected with mBN07 is also Can be in the West The method was identified by the sera of patients who had undergone combined reconstitution, and the monoclonal antibodies that interacted with all four serotypes of dengue fever. This demonstrates that the NS1 line expressed by mBN07 produces immunogenicity. 2: NS1 expressed by mBN07 for the cross-immunogenicity of non-serum type 2 dengue virus and Japanese encephalitis NS1 1. The results of the sero-response test of NS1 expressed by mBN07 on a reconstituted patient have the following possibilities Sex, mBN07-expressed NS1 can be identified by individuals who have previously been infected with dengue virus in reconstituted sera. Serum from 68 individuals with antibodies against dengue virus envelope proteins (by immunoblotting plots against real antigens made from dengue serotypes 1 to 4) was selected for testing control cell extracts infected with mBN07 An immunoblot strip prepared as 32 1336626 with the MVA-GFP-infected cell extract as a control group was allowed to treat the antigen-containing cells in a sample buffer containing no 2-mercaptoethanol and was not heated. Serum for the test of 68 individuals, (91.2%) in the immunoblot map reacted with ΒΝ07 NS1 expressed by mBN07. These serums were further analyzed for the response of all four dengue virus serotypes and NS1 of Japanese encephalitis virus (JEV). The results are shown in Table 1. 54 sera were reacted with all dengue virus serotypes and NS1 of Japanese encephalitis virus (JEV), and 53 of these 54 sera (98. 2%) also reacted with NS1 expressed by mBN07. Seven (7) sera are specific for NS1 of at least one dengue virus serotype and do not react with JEV NS1. The 7 sera also reacted with NS1 expressed by Π1ΒΝ07. The other 7 sera only reacted with NS1 of JEV and did not react with any NS1 of the dengue virus serotype, however 2 of these JEV-specific sera (28.6%) also reacted with NS1 expressed by mBN07. Table 1: BN07 NS1 negative BN07 NS1 positive true DEN NS1 positive 0% (0/7) 100% (7/7) true DEN & JEV NS1 positive 1.85% (1/54) 98.15% (53/54) i positive JEV NS1 positive 71.43% (5/7) 28.57% (2/7) Comparison of antiserum to true NS1 response to NS1 expressed by mBN07. Brackets. The number of samples tested positive / the total number of test samples. Den = dengue, JEV = Japanese encephalitis virus. The same 68 sera were also analyzed for their reactivity to the protein of the prememi3rane. According to the experience of the inventors, the antibody has a higher specificity for the anterior membrane than the antibody for NS1 or E. Therefore, patients who have been infected with dengue will develop antibodies that recognize the dengue virus anterior membrane rather than the pre-jEV membrane, and vice versa. β 33 1336626 along these lines will provide a better prediction of the individual's infection history. . Table 2 shows that serum from 22 individuals reacted with the true dengue pre-membrane protein alone, so 22 patients who had been stunned had only suffered from dengue virus, not JEV infection. These 22 sera all reacted with NS1 expressed by mBN07. Twenty-two patients had previously been confirmed to have been infected with both dengue and JEV. Again, all 22 sera also reacted with NS1 expressed by mBN07. In this series, there were also 21 patients who had previously been confirmed to be infected only with JEV (even if the serum had antibodies that cross-reacted against dengue E). Optionally, one of the 21 JEV responders (82%) reacted with NS1 expressed by mBN07. Only 3 sera in the entire group did not react with either dengue or JEV, and only 1 of these responded to NS1 expressed by mBN07. The most likely reason for this is that the antibody titer is too low to be measured by the immunoblotting method. Table 2: BN07 NS1 negative BN07 NS1 positive true DEN prM positive 0% (0/22) 100% (22/22) true DEN & JEV prM positive 0% (0/22) 100% (22/22) true JEV prM positive 19.0% (5/7) 81.0% (17/21) PrM negative 66.7% (2/3) 33. 3°/〇 (1/3)

Comparison of the response of antiserum to the true anterior membrane to mBN07 NS1. In brackets: The number of samples tested positive / the total number of test samples. DEN = dengue fever, JEV = sputum encephalitis virus. The data in Table 2 also clearly shows that for 6 sera that did not respond to NS1 exhibited by mBN07, 4 were from individuals who had previously been infected with JEV, not dengue fever. The remaining 2 antibodies that did not detect membrane proteins before dengue or JEV 34 1336626 may be too low titers. 2. MBN07-inoculated rabbit and test immunological dot map and ELISA analysis of post-immune serum against dengue virus and Japanese encephalitis virus The rabbits without specific pathogens were immunized subcutaneously according to the inoculation procedure of the present invention as shown below. On the third day, a bottle of sterile-dried vaccine (lxl〇e8 TCID50 BN07 frozen-dried vaccine) was formulated with sterile water to inoculate each of the excipients, and then inoculated again on the 28th day. Take the blood sample before the first inoculation (pre-blood collection) and take it again 10 days after the second inoculation. Day 0 = pre-blood collection, followed by the first vaccination on the 28th day = the second vaccination on the 38th day = blood sampling 2. 1 test pre-blood and anti-cancer serum against the revolutionary cylinder clear type 2 immune ink dot map Figure 5 shows that 1:200 diluted serum was tested in a non-denaturing case, an immunoblot band of dengue 2 virus antigen separated by SDS PAGE and a control strip of uninfected C6/36 cells. band. The results clearly show that when inoculated with mBN07, all rabbits produced a relatively high titer anti-NS1 antibody which cross-reacted with the true NS1 produced by tissue cultured mosquito cells infected with dengue serotype 2. Serum obtained prior to vaccination does not react with any dengue protein on the immunoblot map.

The immunized serum was titrated to 1:1000, 1:2000, 1:4000, 1:10-4, 1:1 (Γ5, 1:10 _6 ' and tested in the absence-denatility case by SDS PAGE The immune dot strip of 35 dengue 2 virus antigens isolated and the control strips of cells not subjected to C6/36. See Figure 6 for the titration of three rabbits to be calculated as 1:10000. Pre-immune and post-immunization The serum was titrated to 1:1〇_2, 1:1〇_3, 1:1〇-4, 1:1〇-5, 1:10_6 ' 1:10'7 and was tested in indirect IgG ELISA The well was coated with dendritic 2 diluted 1:250 and cell lysate of uninfected C6/36. Table 3: 1:102 1:103 1:104 1:105 1:106 i:ur7—Free # 1 Pre -0.012 0. 006 0.007 0.003 -0.002 ο. ooT~ Rabbit #2 Post 0.623 0. 127 0.02 0.004 0.001 ~0.003 Pre -0.012 0 -0.001 -0.003 0 -0. 001 Rabbit #3 Post 0. 402 0.06 -0.007 0.008 -0.003 0. 002 Pre -〇. 008 0. 03 -0. 002 0.005 -0.002 -0.002 ----- Post 0. 907 0. 224 0. 038 0.011-0.001 -0.003 At different dilutions, ELISA and number of sputum in serum of each rabbit before and after immunization (pre = pre-immune Serum, p〇st = serum after immunization. The titration results for each rabbit after immunization are shown in Figure 7. The endpoint titer evaluated for each rabbit immunized with sera is 1:1 〇〇 _2 1_2 Pre-blood and post-cancer serum Serum sequestration of dengue virus serum 1, 3 and 4 plexus and eucalyptus encephalitis virus, 痉 点 图 测 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一 每一The test was performed on a control strip of JE virus antigen + cells not subjected to C6/36 by stripping the dengue 1, 2, 3, 4 of the dengue by SDS PAGE in a non-denaturing condition. Figure 8 shows that each rabbit was immunized with NS1 from dengue serotypes 1, 3 and 4 and reacted with Japanese encephalitis immune dots. 3. Conclusion - Rabbits immunized with mBN07 vaccine were induced to recognize true dengue fever Virus 36 Antibody to serotype 2 NS1. ''A high immunoreactivity was observed when the endpoints in the immunoblot analysis and ELISA were i: i〇_4 and h 10 3 . - The antibody elicited in the rabbit cross-reacted with all other dengue serotypes (1, 3 & 4). The antibody also cross-reacts with NS1 from a heterologous virus such as JEV. Example 3: Growth motility, replication in vivo, and immunological data of the MVA-BN vector virus in selected cell lines. As indicated in the specification section, the DNA of the present invention is preferably inserted into A BN. The concept of a poxvirus or its derivatives. The following examples describe MVa~BN in more detail. The disclosed embodiments allow those skilled in the art to identify MVA_BN and its name. Growth kinetics of L-blister plants: To characterize the MVA-BN, the growth kinetics of this variety were compared with other MVAs that have been delineated. This experiment was carried out by comparing the growth kinetics of the following viruses in the original cells and cell lines listed: MH-BN (virus reserve #23, 18.02.99 untreated, titrated into 2 , 0 X 107 TCIDso/ml); MVA characterized by Mtenburger (US Patent No. 5, 185, 146) 'Alternatively referred to as MVA-HLR; MVA (passage 575) characterized by Anton Mayr (Mayr et al., [1975] Infection 3; 6-14) and additionally referred to as MVA-575 (ECACC V00120707); and 1336626 in the international patent application PCT/EP01/02703 (WO 01/68820) depicting the characteristics of MVA-Vero (virus reserve) ,passage 49,#20,22. 03.99 Untreated, titrated to 4, 2 X 107 TCIDsfl/ml). The original cells and cell lines used were: CEF chicken embryonic fibroblasts (freshly configured from SPF eggs);

HeLa human cervical adenoma (epidermal), ATCC No. CCL-2; 143B human osteosarcoma TK-, ECACC No. 91112502;

HaCaT human keratinocyte cell line, Boukamp et al, 1988, J Cell Biol 106(3): 761-771; BHK baby hamster kidney, ECACC 85011433;

Vero African green monkey kidney cellulose mother cell, ECACC 85020299; CV1 African green monkey kidney fibroblast, ECACC 87032605. In terms of infection, different cells were seeded into 6-well-discs at a concentration of 5 X 105 cells/well at 37 ° C, 5% C 〇 2 in DMEM (Gibco, Cat. No. 61965-026 ) Plus 2% FCS to grow overnight. The cell culture medium was removed and the cells were infected for 1 hour at 37 ° C, 5% C 2 at nearly moi 0.05 (in terms of infection, the number of cells was assumed to double after overnight). The number of cells used for each infection of different cell types was 5 X 1 〇 4 TCID5. And this is called input. Thereafter, the cells were washed three times with DMEM, and finally 1 ml of DMEM, 2% FCS was added and the plate was left at 37 ° C, and incubated at 5% C 2 for 96 hours (4 days). The infection was terminated for titration analysis by freezing the plate at -80 °C. 2. Titration analysis (antibody immunoassay specific to vaccine virus) To quantify the number of viruses 'at the concentration of 1 X 1〇4 cells/well, test cells (CEF) were seeded on 96-well-plate, RPMI (Gibco , Cat. No. 61870-010), 38 1336626 7% FCS, 1% antibiotic/antimycotic (Gibco, Cat. No. 15240-062), and incubated at 37 ° C ' 5% C〇2 Overnight. The 6-well-plate containing the infection experiment was frozen/melted 3 times and diluted 10-1 to ι〇-12 using the RPUI growth medium. The virus dilution was dispensed onto the test cells and at 37. (:, 53⁄4 C〇2 for 5 days to allow CPE (cell pathological effect) to develop. The test cells were fixed (acetone/methanol 1:1) for 10 minutes, washed with PBS and placed at 1:1 于Incubation buffer with multiple strains of vaccinia virus-specific antibodies at room temperature (Quartett

Berlin’Cat. No. 9503-2057) 1 hour. After washing twice with PBS (Gibco, Cat. No, 20012-019), HPR-coupled anti-rabbit antibody (promega Mannheim, Cat. No. W4011) was placed at 1:1000 in incubation buffer. It lasted for 1 hour at room temperature. Wash again twice with pbs and incubate with staining solution (10 ml PBS + 200 μΐ 〇-dimethylhydrazine benzidine saturated solution in 100% ethanol + 15 μΐ freshly prepared Η2〇2) Brown spots (2 hours) were observed. The staining solution was removed and PBS was added to terminate the staining reaction. Each well showing a brown spot indicates positive for CPE and a Kaerber equation to calculate titer (TCID50-based analysis) (Kaerber, G. 1931. Arch. Exp. Pathol. Pharmakol. 162, 480). The virus was used on the one hand to infect a replication group of CEF and BHK with a low repetitive infection, ie 0.05 infected units per cell (5 X 1〇4 TCIDso), which is expected to be tolerant to MVA, and on the other hand , infection of CV-1, Vero, Hela, 143B and HaCat, which is expected to be non-tolerant to MVA. Thereafter, the virus inoculum was removed and the cells were washed three times to remove any residual unabsorbed virus. When the virus extract was prepared, the infection was allowed to continue for 4 days' and then titrated on 39 1336626 CEF cells. Table 1 and Figure 1 show the results of the titration analysis, where the values given are the total amount of virus produced after 4 days of infection. It was shown that all of the diseased kidneys were appropriately expanded in CEF (chicken embryonic fibroblast) cells as expected because this was a cell strain that was tolerant to all MVA. Furthermore, it showed that all viruses were appropriately amplified in BHK (baby hamster kidney cell line) cells. MVA-Vero performed best because BHK is a tolerant cell line. Regarding replication in Vero cells (kidney kidney cell line), MVA-Vero amplifies as appropriate as expected, ie 1000 times higher than the input. Appropriate amplification of MVA-HLR and MVA-575, respectively, increased by more than 33 fold and 1 fold input. Compared to the other 'only MVA-BN, it was found that it was not properly amplified in these cells, i.e., only a 2-fold increase over the input. Further, regarding replication in CV1 cells (kidney kidney cell strain), it was found that MVA-BN was greatly reduced in this cell strain. It shows a decrease of 200~ times lower than the input. MVA-575 also did not amplify to a level above the input and also showed a slightly negative amplification, which is a 16-fold reduction below the input. The MVA-HLR is optimal, with an amplification that is 30-fold higher than the input, followed by MVA-Vero' with a 5-fold increase in the input. The most important thing is to compare the growth kinetics of various viruses in human cell lines. There is an efficient replication in 143B cells (human bone cancer cell lines) which shows that MVA-Vero is the only one showing amplification above input (3-fold increase). All other viruses did not amplify higher than the input, but there was a large difference between MVA-HLR and MVA-BN and MVA-575. MVA-HLR is a wide range of n (less than 1 fold reduction in input), such as MVA-BN showing a minimum reduction (less than 40 300 times the input), followed by MVA-575 (below input 59) Double reduction). In short, MVA-BN is superior in terms of reduction in human 143B cells. Furthermore, regarding replication in HeLa cells (human cervical cancer cells), it was shown that MVA-HLR amplifies well in this cell line, even better than in tolerant BHK cells (Hela = higher than input) A 125-fold increase; BHK = an 88-fold increase over the input), MVA-Vero was also amplified in this cell line (a 27-fold increase over the input). However, MVA-BN and a smaller range of MVA-575 were reduced in these cells (MVA-BN = a 29-fold decrease from the input, while MVA-575 = a 6-fold decrease from the input). Regarding replication in HaCat cells (human keratinocyte cell line), it was shown that MVA-HLR amplifies well in this cell (a 55-fold increase over the input). Both MVA-Vero and MVA-575 showed amplification in this cell line (1.2 and 1.1 fold higher than the input, respectively). However, MVA-BN is the only reduction in presentation (a 5-fold reduction from the input). The results indicate that MVA-BN is the most reduced virus species in this group of viruses: by the human embryonic kidney cells (293: ECACC No. 85120602) showing an amplification ratio of 0.05 to 0.2 (data not incorporated) In Table 1), MVA-BN showed an extreme decrease in human cell lines. Further, it shows an amplification ratio of about 0.0 in 143B cells; an amplification ratio of about 0.04 in HeLa cells; and an amplification ratio of about 0.22 in HaCat cells. Further, the MVA-BN showed an amplification ratio of about 0.0 in CV1. In Vero cells alone, amplification was observed (ratio 2.33), however, when it was in tolerant cell lines such as BHK and CEF, it was not necessarily the same extension (Comparative Table 1). Therefore, it is only known that MVA-BN shows an amplification ratio of less than 1 in all human cell lines (143B, Hela, HaCat, and 293). 41 1336626 Rate 0 MVA-575 shows a similar quantitative curve to MVA-ΒΝ, but not like MVA-BN-like reduction. MVA-HLR amplifies well in all tested cells (except for 143] B cells). Therefore, it is considered to be sufficient for replication in all tested cell lines (except for 14 cells). In some cases, its amplification in human cell lines (HeLa) is even better than in tolerant cell lines (BHK). "MVA-Vero did show amplification in all cell lines, however it was smaller than the range shown by MVA-HLR (ignoring the results of 143B). However, in terms of reduction, it cannot be considered to be the same as MVA-BN or MVA-575. "Reproduction in vivo. If some MVA varieties are clearly replicated in vivo, for different MVA Reproduction of the cultivar in vivo can be detected by using the genetically engineered mouse model AGR129. This mouse cultivar has been disrupted in the IFN receptor type I (IFN-α/β) and type II (IFN-γ) genes and RAG. The gene. Due to such damage, the mouse has no IFN-based filling and is unable to produce mature 8 and T cells, so it is severely immune to abandonment and is very resistant to replication. MVA-BN, MVA-HLR or MVA572 at 10〇7pfu To immunize (i, p.) six groups of mice (used in Germany on 120, sputum individuals) and monitor clinical symptoms daily. All mice vaccinated with MVA HLR or MVA 572 died within 28 to 60 days respectively. In the necropsy test, there is a common phenomenon of serious viral infection in the main organs, and MVA (l〇8pfu) can be found again from the ovary by standard plaque analysis. Conversely, the same dose of MVA-BNC corresponds. For the registered variety ECACC V 00083008) Rats after vaccination survived for more than 90 days and were not re-discovered from organs or tissues without MVA. 42 1336626 When data on in vitro and in vivo were obtained together, studies have shown that MVA-BN is more than maternal and commercial The MVA_HLR variety is more severely reduced. 4. The MVA of different varieties is also different in stimulating immune response. The vaccination of a sufficient variety of vaccinia induces an immune response in mice, while the high dose is fatal. Although the MVA is highly reduced And there is a - reducing ability in the replication of mammalian cells, however, there is a difference in the reduction of MVA between different varieties. Indeed, MVA BN has more reduction than other MVA varieties' even the parent variety MVA 575. To determine whether differences in reduction would affect the efficacy of MVA in inducing a protective immune response, different doses of MVA BN and MVA 575 were compared in a lethal vaccinia stimulation model. The level of protection was measured by stimulation for 4 days. After that, the titer determined in the ovarian vaccinia is reduced, and this allows quantitative evaluation of MVA of different doses and different varieties. The lethal stimulation model MVA BN or MVA 575 at different doses (102, 104 or 〇6TCID5D/ml) immunized (ip) 6-8-week-old females with specific pathogen-free (n=5). MVA-BN and MVA-575 were CEF cells were propagated and purified by sucrose and formulated into 7.4 in Tris. After three weeks, the rats received an increase in the same dose of MVA and MVA varieties, followed by a vaccinated variety of vaccinia two weeks later. Lethal stimulation (i_pj. The WR-L929 TK+ or IHD-J variety can be used as a fully replicated vaccinia virus (abbreviated “rVV”). Rats in the control group received a placebo. The protective effect can be measured by the amount of decrease in titer determined in the ovary by standard spot analysis after 4 days of stimulation. Here, mice were sacrificed on the 4th day after stimulation, and the ovaries were removed, homogenized in PBS (1 ml) and VERO cells were used to determine viral titer by standard spot analysis (Thomson et al., 1998, 43 1336626).

Immunol. 160: 1717) ° After 4 days of stimulation, with a 100% reduction in ovarian rVV titers, 10 or 10 TCIDso/ml of MVA-BN or MVA-575 immunized mouse strains Fully protected (Figure 2). The stimulating virus was cleared. However, it can be observed at low doses that the protection provided by MVA-BN or MVA-575 is different. MVA 575 mice immunized with lOlCH^/ml were unprotected and were evaluated with high ovarian rVV titers (mean 3.7xl 〇 7pfu +/- 2.11 χίο7). In contrast, mice vaccinated with the same dose of MVA-BN induced a significant reduction (96%) in terms of high ovarian rVV titers (mean 〇 21 x l 〇 7 pfu +/- 〇 287 x 〇 7). Control group mice receiving placebo had an average viral titer of 5·11 xl07pfu (+/- 3.59 xlO7) (Figure 2). Both MVA strains induced a protective immune response against lethal Γνν stimulation in mice. Although the efficacy of the two MVA strains at the higher doses is comparable, the efficacy of the two MVA strains is significantly different when compared to the optimal dose. On the protective immune response induced to combat lethal rVV stimulation, the intensity of MVA-BN is greater than that of its parental variety MVA-575', which is clearly related to the decrease in the increase in MVA-575 and MVA-BN. 4. MVA-BN in the first-push-inoculation therapy 5. 1. After inoculation of mice with different smallpox vaccines, antibody production against mva compares the efficacy of MVA-BN with other MVA and the previously used pox strains used in the eradication of smallpox. These include the use of the Elstree and Wyeth vaccinia varieties produced by CEF and transmitted through the tail cuts and the single immunization of MVA 572 used in Germany before eradication. In addition, before the vaccines of both MVA-BN and MVA 572 were compared, the cuts were followed by Elstree. Each group used 44 1336626 with 8 BALB/C mice, and all MVAClxlO7 TCID5d) were administered subcutaneously at weeks 0 and 3. Two weeks after the immunization was boosted, the mice were stimulated with vaccinia (IHD-J) and the titer in the ovaries was measured 4 days after the stimulation. All vaccines and treatments cause 100% protection. The immune response elicited using these different vaccines or therapies is measured prior to stimulation in the animals. Neutralizing antibodies, sputum cell proliferation, cytokine products (IFN-γ and IL-4) and IFN-γ assays produced by sputum cells were used. The level of sputum cell response induced by MVA-BN is generally equivalent to other MVA and vaccinia virus when measured by EL I spot, showing bio-equivalent. After different vaccination therapies, the weekly titer of antibodies against MVA showed that the rate and size of the antibody were significantly increased by MVA-BN vaccination compared to other inoculation methods (pn). Indeed, compared to mice vaccinated with MVA 572, the antibody titer against MVA was significantly higher at 2, 4, and 5 weeks (1 week after the 4th week of boost) when vaccinated with MVA-BN ( ρ>0· 05). After the booster vaccination at week 4, the antibody titer in the MVA-BN population was also significantly higher than the single vaccination with the vaccinia strain Elstree or Wyeth. These results clearly show that two of the MVA_BN vaccinates induced a higher antibody response compared to the typical single vaccination with traditional bovine cancer strains (Elstree and Wyeth), and confirmed this finding from the region 丨5, MVA - more immunogenic than other MVA strains. 5.2. In the influenza stimuli model, the initial and push-up therapy produces the same level of protection as the DNA-first MVA-push therapy, which will be used to generate a highly active CTL response for the first time. It has been reported as the best DMA initial/MVA push therapy comparison. Different therapies were evaluated using a murine polyhedral structure encoded by (10) eight vectors or MVA-BN and the CTL induced levels were compared at 45 1336626 ELISPOT, while the measured reactivity was measured as influenza-stimulated The extent of protection provided. Results The DNA quality of the murine polyhedra (10 CTL epitope, including influenza, ovalbumin) has been previously described (Th〇ms〇n et al, i998,] Imnmnol. 160: 1717). This murine polyhedron was inserted into the deletion position II of MVA BN, grown in CEF cells, stored in sucrose and formulated in Tris to form a 讪7·4 〇 vaccination step in the current study, using a specific virus for 6-8 weeks. Large female BALB/c (H-2d) mice. Five mouse populations were used for Eusp〇T analysis and 6 mice per group were used for influenza stimulation experiments. The mice were inoculated with MVA or DNA encoding a murine polyhedron as detailed in the results, using different initial _ push-ups. In the case of DNA vaccination, the mice were anesthetized and then under anesthesia, 50 gg of endotoxin-free plastid DNA (in 50 μM of pbs) was injected into the quadriceps. The primary immunization used was performed by intravenously administering 1〇7 pfu of MVA-BN per mouse or subcutaneously administering 107 pfu or i〇8 pfu of mva-BN per mouse. Push-up immunity was given 3 weeks after the initial immunization. The push-up of plastid DNA is carried out in the same manner as the primary immunization using DNA (see above). In order to establish a CTL response, the influenza CTL epitope peptide (TYQRTRALV), P. Berghei epitope peptide (SYIPSAEKI), giant cell virus peptide epitope (YPHFMPTNL) and/or LCV are used. Two weeks after the final boosting of the peptide epitope (RPQASGVYM), a standard ELISPOT assay was performed on splenocytes (Schneider et al., 1998, Nat. Med. 4; 46 1336626 397-402). In the stimulation experiment, the mice were anesthetized, and the mice were infected with a sub-lethal dose of the commonly used influenza virus, Mem71 (4.5 x lO 5 pfu in 50 ml of PBS) i.n. On day 5 post-infection, the lungs were removed and the virus titer in duplicate was determined in the Mad i n-Darby dog tubular cell line using standard epidemic plaque assay. result:

The DNA vaccine alone was used, and the CTL line induced by the 4H-2 d epitope encoded by the mouse polyhedron was insufficient, and only the epitope of P. Berghei (SYIPSAEKI) and the lymphatic plexus meningeal virus (RPQASGVYM) were only available. A small amount of reaction was detected. Conversely, the use of DNA initial MVA push-up therapy (subcutaneous administration of 107 pfu MVA-BN) resulted in significantly higher CTL induction, for SLY (8-fold increase) versus RPQ (3-fold increase), and A response to the third epitope of the murine giant cell virus (YPHFMPTNL) was observed (Fig. 3A). However, subcutaneous administration of 107ρίϋΜνΐβΜ in the same initial push-up therapy induces the same reaction as the DNA using MVA-BN (Fig. 3). Surprisingly, when immunized with an MVA-BN (107 TCID50), there was no significant difference in the number of CTLs induced by the three epitopes, indicating that the second immunization did not significantly increase CTL. reaction. It has previously been shown that subcutaneous administration of 1〇7 pfu MVA is the most inefficient route and virus concentration when inoculated with MVA from other strains, especially when compared to intravenous immunization (Schneider et al. 1998). In order to define the optimal immunotherapy, the above experiments were repeated to change the number of viruses or change the mode of administration. In one experiment, 107 pfu of the MVA-BN vaccine was administered intravenously (Fig. 3B). In another experiment, 108 pfu of MVA-BN was administered subcutaneously (Fig. 3C). 47 1336626 In these experiments, the MVA-ΒΝ initial-push-immunization induced a higher average number of CTLs for all three CTL epitopes than the initial MVA push-up therapy for DNA. Furthermore, unlike subcutaneous administration of 1〇7 pfu of MVA-BN, immunization with intravenous administration of 107 pfu of MVA-BN and subcutaneous administration of 1〇8 pfu significantly increased the CTL response. This clearly indicates that MVA-BN can be used to increase the CTL response in the presence of immunity to the vector in advance. references

Nimmannitya S, Kalayaranaro S, Nisalak A and Innes B. 1990. The second episode of dengue hemorrhagic fever. Southeast Asian Journal of Tropical Medicine and Public Health, 21:699

Burke DS and Monath TP. 2001, Flavivirus. The Virus, Fourth Edition, edited by David M Knipe and Peter M Howley. By Lippincott

Published by Williams and Wilkins, Philadelphia. Page 1043-1 125

Flamand Μ, Megret F, Mathieu Μ, LePault, Rey FA, and Deubel V., 1999. The dengue virus type 1 non-structural glycoprotein NS1 is secreted by mammalian cells and is a soluble hexamer in the glycosylation-dependent form. J. Virol., 73:6104-6110

Jang SK 'Davies MV, Kaufman RJ and Wimmer E., 1989. By in vivo, the ribosome enters the 5, non-translated region of the encephalomyocarditis virus RNA to initiate protein synthesis. J. Virol., 63:1651-60 48 1336626

Lindenbach BD and Rice CM., 2001, flavivirus and its replication. In the field of viruses, the fourth edition. Edited by David M Knipe and Peter M Howley.

Lippincott Williams and Wilkins, Philadelphia. Page 991-1041

Schlesinger JJ, Brandriss MW and Walsh EE., 1987. The mice were protected from dengue 2 viral encephalitis by immunization with the dengue 2 virus non-structural protein NS1. J. Gen. Virol., 68: 853-7 Schesinger JJ, Foltzer Μ and Chapman S., 1993. For the yellow fever virus NS1, the Fc portion of the antibody protects the mouse from the decisive site of yellow fever encephalitis. Virology. 192: 132-41 The techniques and procedures described herein are familiar to molecular organisms and viruses, particularly those familiar with the genetic manipulation of flaviviruses and poxviruses. The details of the techniques and procedures described can be found in the following literature sources:

Molecular selection, laboratory manual. second edition. Published by J. Sambrook, E. F. Fritsch and T. Maniatis. Cold Spring Harbor Laboratory Press. 1989 Handbook of Virology Methods. Edited by Brian WJ Mahy and Hi 1 lar 0 Kangro. Academic Press. 1996 Molecular Virology · Practical Introduction. Edited by AJ Davison and RM Elliott. 49 1336626 Practical entry series. IRL Press at Oxford University Press.

Oxford 1993. Chapter 9: Gene Expression of Vaccinia Virus Vectors Current rules for molecular biology. Issuer: John Wiley and Son Inc. 1998. Chapter 16, Section IV: Performance of proteins used in bovine cancer virus-borne animals. Antibody, laboratory manual. Edited by Har 1 ow and David Lane. Published by Cold Spring Harbor Laboratory, 1988. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a diagram showing a dengue NGC variety "signal sequence + NSl" cDNA protein coding sequence as an embodiment of the present invention. The beginning of the NS1 gene in the natural text is indicated by an arrow. An important feature is the addition of an ATG start codon and a stop codon ("TAG") in this example. The number of nucleotide sequences refers to the position of the NGC genomic genome (Gene Database Accession No. AF038403). The nucleotide in Figure 1A corresponds to the amino acid of SEQ ID NO: 5. The amino acid is shown separately in SEQ ID NO: 6. Figure 1B: A graph of the plastid PAF7NS1 containing the protein coding sequence for the dengue NGC variety "signal sequence + NS1". Figure 1C: The nucleotide sequence of the NS1 cassette in plastid pAF7 shows that the promoter linkage position of this cassette was PCR amplified with OBN345 and oBN338. The nucleotide and amino acid sequence in Figure 1C corresponds to SEQ ID NO: 7. The amino acid is shown separately as SEQ ID NO: 8. Figure 1D: Top: Kyte-Doolittle hydrophilic dot map of the NS1 amino acid sequence of the dengue NGC variety (dengue NCG polyprotein gene library Amino acid 776 to 1127 of accession number AF038403). The value is higher than zero = hydrophobic. 50 1336626 B: Kyte-Doolittle hydrophilic dot map (amino acid 748 to 775) of the dengue NCG NS1 amino acid sequence containing the signal sequence of the last 28 amino acids derived from the C-terminus of the E protein. The entire amino acid sequence represents the amino acid 748 to 1127 of the dengue NCG polyprotein (Gene Database Accession No. AF038403), which starts with the "ATG" start codon but lacks a stop codon. Sig = signal sequence. The value is higher than zero = hydrophobic. Fig. 2··"pox virus promoter + signal sequence + NS1M shows the nucleotide sequence of the cardinal. The nucleotide and amino acid sequences in Figure 2 correspond to SEQ ID NO: 9. The amino acid is shown separately in SEQ ID NO: 10. Briefly, the minimal/pox virus early/late promoter element controls the expression of the NS1 protein of dengue virus serotype 2, wherein the N-terminus of the NS1 protein is fused to the 28 C-terminal amino acids of the E-protein. The translation terminates in the TAG stop codon in the inserted nucleic acid sequence. Figure 3A: The NS1 expression cassette was cloned into the Xh〇 I position of the flat end of pBNX07 (blunt end blunt end) to generate the selected strain pBN41. PPr = poxvirus promoter, D2F1 = side 2 of deletion 2, NPT II = neomycin resistance gene, IRES = ribosome binding site, EGFP = elevated green fluorescent protein, NS1 (in PBN41) = signal Sequence + NS1, D2F2 = side 2 of deletion 2, Sig = signal sequence. AmpR = ampicillin resistance gene. Figure 3B: Hind III map of MVA (Gene Library U94848) showing six missing addresses of MVA (-J- = deleted junction). "PPr+NPT II + IRES+EGFP+PPr+NSl" The cassette is inserted into the missing 2 position of MVA. PPr = poxvirus promoter, NPT II dimycin resistance gene (protein coding sequence), IRES = ribosome binding site and NS1 = signal sequence + dengue 2 NGC species NS1 protein coding sequence. 51 1336626 Figure 4: Immunoblot map showing mBN07 and true dengue virus serotype 2 with uninfected cell control group, probed with monoclonal antibodies against dengue virus NS1 protein. The arrow on the left refers to the natural NS1 dimer, and the arrow on the right refers to the NS1 monomer, which is viewed after the sample has boiled. 2ME = 2 mercaptoethanol. Figure 5: Serum diluted to 1:200 was tested on a strip of immune dots of dengue virus serotype 2 antigen isolated by SDS PAGE without reduction and without heating, and uninfected C6 /36 cells on the control strip. Band 1 = dengue virus serotype 2 antigen band; band 2 = control band; a = pre-immune serum, and b = post-epidemic serum. Figure 6: Serum after immunization was titrated to 1:1000, 1:2000, 1:4000, 1:10_4, 1:1 (Γ5, 1:10_6, and tested in the absence of reduction and no heating, Isolation of the dengue 2 virus antigen on the control strip after SDS PAGE and on the control strip of uninfected C6/36 cells. Note: For rabbits #1 and #2, the NS1 band diluted 10_4 cannot be scanned. Observed on the photograph, however, the actual band can be observed by the naked eye. 1 = dengue virus serotype 2 antigen band, 2 = control band, A = rabbit #1, B = rabbit #2, and C = rabbit# 3. Figure 7: Point of ELISA absorption reading for serum titration after immunization of all three exons. Figure 8: Rabbit serum diluted to 1:1000 each was tested, without reduction and On heating, strips of dengue 1, 2, 3, 4+JE virus antigens on SDS PAGE and 52 1336626 control strips on uninfected C6/36 cells without heating. Note · For The MSI band with rabbit #2 serum dengue 3 and 4 can not be observed on scanned photos, but the actual band can be Observed by the naked eye. 1 = dengue virus serotype 1 antigen band, 2 = dengue virus serotype 3 antigen band, 3 = dengue virus serotype 4 antigen, 4 = JE virus antigen band, and 5 = control group. [Main component symbol description] (none)

53 1336626 Sequence Listing

<110> Bavarian Nordic A/S

Venture Technologies Sdn Bhd <12〇> Flavivirus NS1 subunit vaccine <130> NS1 dengue <140> 0000 <141> 2001-12-04 <160> 10 <170> Patentln Ver. 2.1

<210> 1 <211> 30 <212> DNA <213> Synthetic sequence <220><223> Description of Artificial Sequence: primer <400> 1 30 ttgttagcag ccggatcgta gacttaatta <210> 2 <211> 62 <212> DNA <213> Synthetic sequence

<220><223> Description of Artificial Sequence:primer <400> 2 caaaaaattg aaattttatt tttttttttt ggaatataaa taaaaacacg ataataccat 60 QQ 62 <210> 3 <211> 33 <212> DNA <213> ^ <220><223> Description of Artificial Sequence: primer 1 1336626 <400> 3 aatagatctc tactaggctg tgaccaagga gtt 33

<210> 4 <211> 33 <212> DNA <213 > Synthetic sequence Sequence <220><223> Description of synthetic sequence: primer. <400> 4 acaagatctg gaatgaattc acgtagcacc tea 33

<210> 5 <211> 1143 <212> DNA <213> Dengue Virus Noodle 2 <220><221> CDS <222> (1) . (1140) <223> E protein Signal sequence + NS1 code sequence <400> 5 atg aat tea ege age acc tea ctg tet gtg tea eta gta ttg gtg gga 48

Met Asn Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly 1 5 10 15 gtc gtg aeg ctg tat ttg gga gtt atg gtg cag gee gat agt ggt tgc 96

Val Val Thr Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys 20 25 30 gtt gtg age tgg aaa aac aaa gaa ctg aag tgt ggc agt ggg att ttc 144

Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser Gly lie Phe 35 40 45 ate aca gac aac gtg cac aca tgg aca gaa caa tac aag ttc caa cca 192 lie Thr Asp Asn Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Pro 50 55 60 gaa tee cct tea aag eta get tea get ate cag aaa get cat gaa gag 240

Glu Ser Pro Ser Lys Leu Ala Ser Ala lie Gin Lys Ala His Glu Glu 65 7 0 75 80 ggc att tgt gga ate ege tea gta aca aga ctg gaa aat ctg atg tgg 288 Giy lie Cys Gly lie Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp 85 90 95 aaa caa ata aca cca gaa ttg aat cac att eta tea gaa aat gag gtg 336 Lys Gin He Thr Pro Glu Leu Asn His lie Leu Ser Glu Asn Glu Val 100 105 110 aag ttg act att atg aca gga Gac ate aaa gga ate atg cag gca gga 384 Lys Leu Thr lie Met Thr Gly Asp lie Lys Gly lie Met Gin Ala Gly 115 120 125 aaa ega tet ctg cag ccc cag ccc act gag ctg aag tat tea tgg aaa 432 Lys Arg Ser Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys 130 135 140 aca tgg ggc aaa geg aaa atg etc tet aca gag tet cat aac cag acc 480 Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr 145 150 155 160 ttt etc att gat ggc ccc gaa aca gca gaa tgc ccc aac aca aac aga 528 Phe Leu lie Asp Gly Pro Glu Thr Ala Glu Cys P Ro Asn Thr Asn Arg 165 170 175 get tgg aat teg ctg gaa gtt gaa gac tat ggc ttt gga gta ttc acc 576 Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr 180 185 190 acc aat ata tgg eta aag Ttg aga gaa aag cag gat gta ttc tgc gac 624 Thr Asn lie Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp 195 200 205 tea aaa etc atg tea geg gee ata aaa gac aac aga gee gtc cat gee 672 Ser Lys Leu Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala 210 215 220 gat atg ggt tat tgg ata gaa agt gca etc aat gac aca tgg aag ata 720 Asp Met Gly Tyr Trp He Glu Ser Ala Leu Asn Asp Thr Trp Lys lie 225 230 235 240 gag aaa gee tet ttc ate gaa gtt aaa age tgc cac tgg cca aag tea 768 Glu Lys Ala Ser Phe lie Glu Val Lys Ser Cys His Trp Pro Lys Ser 245 250 255 cac acc etc tgg agt aat gga gtg tta gaa agt Gag atg ata att cca 816 His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie lie Pro 26 0 265 270 I3J6626 aag aat ttc get gga cca gtg tea caa cac aac tac aga cca ggc tac 864 Lys Asn Phe Ala Gly Pro Val Ser Gin His Asn Tyr Arg Pro Gly Tyr 275 280 282 cat aca caa aca gca gga cca tgg cat eta Ggt aag ett gag atg gac 912 His Thr Gin Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp 290 295 300 ttt gat ttc tgc gaa gga acc aca gtg gtg gtg act gag gac tgt gga 960 Phe Asp Phe Cys Glu Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly 305 310 315 320 aat aga gga ccc tet tta aga aca act act gcc tet gga aaa etc ata 1008 Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu He 325 330 335 aca Gaa tgg tgc tgc ega tet tgc aca tta cca ccg eta aga tac aga 1056 Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg 340 345 350 ggt gag gac gga tgc tgg tac ggg atg gaa ate aga cca ttg aaa Gag 1104 Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu lie Arg Pro Leu Lys Glu 355 360 365 Aaa gaa gag aat ttg gtc aac tcc ttg gtc aca gcc tag 1143 Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala 370 375 380

<210> 6 <211> 380 <212> PRT

<213> Dengue virus type 2 <400> 6

Met Asn Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly 1 5 10 15

Val Val Thr Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys 20 25 30

Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser Gly lie Phe 35 40 45 lie Thr Asp Asn Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Pro 50 55 60

Glu Ser Pro Ser Lys Leu Ala Ser Ala lie Gin Lys Ala His Glu Glu 4 1336626 65 70 75 80 Gly He Cys Gly lie Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp 85 90 95 Lys Gin lie Thr Pro Glu Leu Asn His Lie Leu Ser Glu Asn Glu Val 100 105 no Lys Leu Thr lie Met Thr Gly Asp lie Lys Gly lie Met Gin Ala Gly 115 120 125 Lys Arg 130 Ser Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys 135 140 Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr 145 150 155 160 Phe Leu lie Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg 165 170 175 Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr 180 185 190 Thr Asn lie Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp 195 200 205 Ser Lys Leu Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala 210 215 220 Asp Met Gly Tyr Trp lie Glu Ser Ala Leu Asn Asp Thr Trp Lys lie 225 230 235 240 Glu Lys Ala Ser Phe work le Glu Val Lys Ser Cys His Trp Pro Lys Ser 245 250 255 His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie li e Pro 260 265 270 Lys Asn Phe Ala Gly Pro Val Ser Gin His Asn Tyr Arg Pro Gly Tyr 275 280 285 His Thr Gin Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp 290 295 300 Phe Asp Phe Cys Glu Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly 305 310 315 320 Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu lie 5 1336626 a 325 330 335

Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg 340 345 350

Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu lie Arg Pro Leu Lys Glu 355 360 365

Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala 370 375 380 <210> 7 <211> 1296 <212> DNA <213> Dengue Fever <220>

<221> CDS <222> (50) · (1189) <220><221> Introduction_Combination 5. <222> (12) . . (32) <220><221>引__合合<222> (1243) . . (1273) <400> 7 ttttcctttg aaaaacacga taataccatg ggaattcccc cgatctgga atg aat tea

Met Asn Ser 1 ege age acc tea ctg tet gtg tea eta gta ttg gtg gga gtc gtg aeg

Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly Val Val Thr 5 10 15 ctg tat ttg gga gtt atg gtg cag gcc gat agt ggt tgc gtt gtg age

Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys Val Val Ser 20 25 30 35 tgg aaa aac aaa gaa ctg aag tgt ggc agt ggg att ttc ate aca gac

Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser Gly lie Phe lie Thr Asp 40 45 50 aac gtg cac aca tgg aca gaa caa tac aag ttc caa cca gaa tcc cct 58 106 154 202 250 298 1336626

Asn Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Pro Glu Ser Pro 55 60 65 tea aag eta get tea get ate cag aaa get cat gaa gag ggc att tgt Ser Lys Leu Ala Ser Ala lie Gin Lys Ala His Glu Glu Gly lie Cys 70 75 80 346 gga ate ege tea gta aca aga ctg gaa aat ctg atg tgg aaa caa ata Gly lie Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gin lie 85 90 95 394 aca cca gaa ttg aat cac att eta tea Gaa aat gag gtg aag ttg act Thr Pro Glu Leu Asn His lie Leu Ser Glu Asn Glu Val Lys Leu Thr 100 105 110 115

Att atg aca gga lie Met Thr Gly ctg cag ccc cag Leu Gin Pro Gin 135 gac ate aaa gga ate atg Asp lie Lys Gly lie Met 120 125 ccc act gag ctg aag tat Pro Thr Glu Leu Lys Tyr 140 cag gca gga aaa ega tet Gin Ala Gly Lys Arg Ser 130 tea tgg aaa aca tgg ggc Ser Trp Lys Thr Trp Gly 145 442 490 aaa geg aaa atg etc tet aca gag tet cat aac cag acc ttt etc att Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr Phe Leu lie 150 155 160 gat ggc ccc gaa aca gca gaa tgc ccc aac aca aac aga get tgg aat Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn 165 170 175 538 586 teg ctg gaa gtt gaa gac Tat ggc ttt gga gta ttc acc acc aat ata Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn lie 180 185 190 195 634 tgg eta aag ttg aga gaa aag cag gat gta ttc tgc gac tea aaa etc Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp Ser Lys Leu 200 205 210 atg tea geg gee ata aaa gac aac aga gee gtc cat gee gat atg ggt Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala Asp Met Gly 215 220225 682 730 tat tgg ata gaa agt gca etc aat gac aca tgg aag ata gag aaa gee Tyr Trp lie Glu Ser Ala Leu Asn Asp Thr Trp Lys lie Glu Lys Ala 230 235 240 778 tet ttc ate gaa gtt aaa age tgc cac tgg cca Aag tea cac acc etc 826 7 1336626

Ser Phe lie Glu Val Lys Ser Cys His Trp Pro Lys Ser His Thr Leu 245 250 255 tgg agt aat gga gtg tta gaa agt gag atg ata att cca aag aat ttc 874

Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie lie Pro Lys Asn Phe 260 265 270 275 get gga cca gtg tea caa cac aac tac aga cca ggc tac cat aca caa 922

Ala Gly Pro Val Ser Gin His As Tyr Arg Pro Gly Tyr His Thr Gin 280 285 290 aca gca gga cca tgg cat eta ggt aag ett gag atg gac ttt gat ttc 970

Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe 295 300 305 tgc gaa gga acc aca gtg gtg gtg act gag gac tgt gga aat aga gga 1018

Cys Glu Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly 310 315 320 ecc tet tta aga aca act act gcc tet gga aaa etc ata aca gaa tgg 1066

Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu lie Thr Glu Trp 325 330 335 tgc tgc ega tet tgc aca tta cca ccg eta aga tac aga ggt gag gac 1114

Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp 340 345 350 355 gga tgc tgg tac ggg atg gaa ate aga cca ttg aaa gag aaa gaa gag 1162

Gly Cys Trp Tyr Gly Met Glu lie Arg Pro Leu Lys Glu Lys Glu Glu 360 365 370

Aat ttg gtc aac tcc ttg gtc aca gcc tagtagggat cgggggagct 1209

Asn Leu Val Asn Ser Leu Val Thr Ala 375 380 cactagtgga tccctccagc tegagaggne taattaatta agtetaegat ccggctgcta 1269 acaaagcccg aaaggaaget gagttgg 1296

<210> 8 <211> 380 <212> PRT < 213 > dengue virus type 2 <400>

Met Asn Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly 15 10 15 8 1336626

Val Val Thr Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys 20 25 30

Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser Gly lie Phe 35 40 45 lie Thr Asp Asn Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Pro 50 55 60

Glu Ser Pro Ser Lys Leu Ala Ser Ala lie Gin Lys Ala His Glu Glu 65 70 75 80

Gly lie Cys Gly lie Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp 85 90 95

Lys Gin lie Thr Pro Glu Leu Asn His lie Leu Ser Glu Asn Glu Val 100 105 no

Lys Leu Thr lie Met Thr Gly Asp He Lys Gly He Met Gin Ala Gly 115 120 125

Lys Arg Ser Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys 130 135 140

Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr 145 150 155 160

Phe Leu lie Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg 165 170 175

Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr 180 185 190

Thr Asn lie Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp 195 200 205

Ser Lys Leu Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala 210 215 220

Asp Met Gly Tyr Trp lie Glu Ser Ala Leu Asn Asp Thr Trp Lys lie 225 230 235 240

Glu Lys Ala Ser Phe lie Glu Val Lys Ser Cys His Trp Pro Lys Ser 245 250 255

His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie lie Pro 260 265 270 9 1336626

Lys Asn Phe Ala Gly Pro Val Ser Gin His As Tyr Arg Pro Gly Tyr 275 280 285

His Thr Gin Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp 290 295 300

Phe Asp Phe Cys Glu Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly 305 310 315 320

Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu lie 325 330 335

Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg 340 345 350

Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu He Arg Pro Leu Lys Glu 355 360 365

Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala 370 375 380

<210> 9 <211> 1227 <212> DNA <213> Dengue virus type 2 <220><221> promoter <222> (5).. (48}

<223> minimal poxvirus promoter element <220>

<221> CDS <222> (85) (1224) <223> NS1 <400> 9 ctcgacaaaa aattgaaatt ttattttttt tttttggaat ataaataaaa acacgataat 60 accatgggaa ttccccgatc tgga atg aat tea ege age acc tea ctg tet 111

Met Asn Ser Arg Ser Thr Ser Leu Ser 1 5 gtg tea eta gta ttg gtg gga gtc gtg aeg ctg tat ttg gga gtt atg 159

Val Ser Leu Val Leu Val Gly Val Val Thr Leu Tyr Leu Gly Val Met 10 10 15 20 25 gtg cag gcc gat agt ggt tgc gtt gtg age tgg aaa aac aaa gaa ctg

Val Gin Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu 30 35 40 aag tgt ggc agt ggg att ttc ate aca gac aac gtg cac aca tgg aca

Lys Cys Gly Ser Gly lie Phe lie Thr Asp Asn Val His Thr Trp Thr 45 50 55 gaa caa tac aag ttc caa cca gaa tcc cct tea aag eta get tea get

Glu Gin Tyr Lys Phe Gin Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala 60 65 70 ate cag aaa get cat gaa gag ggc att tgt gga ate ege tea gta aca lie Gin Lys Ala His Glu Glu Gly lie Cys Gly lie Arg Ser Val Thr 75 80 85 aga ctg gaa aat ctg atg tgg aaa caa ata aca cca gaa ttg aat cac

Arg Leu Glu Asn Leu Met Trp Lys Gin lie Thr Pro Glu Leu Asn His 90 95 100 105 att eta tea gaa aat gag gtg aag ttg act att atg aca gga gac ate lie Leu Ser Glu Asn Glu Val Lys Leu Thr lie Met Thr Gly Asp lie 110 115 120 aaa gga ate atg cag gca gga aaa ega tet ctg cag ccc cag ccc act

Lys Gly lie Met Gin Ala Gly Lys Arg Ser Leu Gin Pro Gin Pro Thr 125 130 135 gag ctg aag tat tea tgg aaa aca tgg ggc aaa geg aaa atg etc tet

Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser 140 145 150 aca gag tet cat aac cag acc ttt etc att gat ggc ccc gaa aca gca

Thr Glu Ser His Asa Gin Thr Phe Leu lie Asp Gly Pro Glu Thr Ala 155 160 165 gaa tgc ccc aac aca aac aga get tgg aat teg ctg gaa gtt gaa gac

Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp 170 175 180 185 tat ggc ttt gga gta ttc acc acc aat ata tgg eta aag ttg aga gaa

Tyr Gly Phe Gly Val Phe Thr Thr Asn lie Trp Leu Lys Leu Arg Glu 190 195 200 aag cag gat gta ttc tgc gac tea aaa etc atg tea geg gee ata aaa

Lys Gin Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala lie Lys 11 1336626 205 210 215 gac aac aga gee gtc cat gee gat atg ggt tat tag ata gaa agt gca 783 Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Lie Glu Ser Ala 220 225 230 etc aat gac aca tgg aag ata gag aaa gee tet ttc ate gaa gtt aaa 831 Leu Asn Asp Thr Trp Lys lie Glu Lys Ala Ser Phe lie Glu Val Lys 235 240 245 age tgc cac tgg cca aag tea Cac acc etc tgg agt aat gga gtg tta 879 Ser Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu 250 255 260 265 gaa agt gag atg ata cca aag aat ttc get gga cca gtg tea caa 927 Glu Ser Glu Met lie lie Pro Lys Asn Phe Ala Gly Pro Val Ser Gin 270 275 280 cac aac tac aga cca ggc tac cat aca caa aca gca gga cca tgg cat 975 His Asn Tyr Arg Pro Gly Tyr His Thr Gin Thr Ala Gly Pro Trp His 285 290 295 eta ggt aag ett gag atg gac ttt gat ttc tgc gaa gga acc aca gtg 1023 Leu Gly Lys Leu Gl u Met Asp Phe Asp Phe Cys Glu Gly Thr Thr Val 300 305 310 gtg gtg act gag gac tgt gga aat aga gga ccc tet tta aga aca act 1071 Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr 315 320 325 act gee tet gga aaa etc ata aca gaa tgg tgc tgc ega tet tgc aca 1119 Thr Ala Ser Gly Lys Leu lie Thr Glu Trp Cys Cys Arg Ser Cys Thr 330 335 340 345 tta cca ccg eta aga tac aga ggt gag gac gga tgc Tgg tac ggg atg 1167 Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met 350 355 360 gaa ate aga cca ttg aaa gag aaa gaa gag aat ttg gtc aac tee ttg 1215 Glu lie Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu 365 370 375 gtc aca gee tag 1227 Val Thr Ala 380

<210> 10 12 1336626

<211> 380 <212> PRT <213> Dengue virus type 2 <400>

Met Asn Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly 15 10 15

Val Val Thr Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys 20 25 30

Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser Gly lie Phe 35 40 45 lie Thr Asp Asn Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Pro 50 55 60

Glu Ser Pro Ser Lys Leu Ala Ser Ala lie Gin Lys Ala His Glu Glu 65 70 75 80

Gly lie Cys Gly lie Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp 85 90 95

Lys Gin lie Thr Pro Glu Leu Asn His work Leu Ser Glu Asn Glu Val 100 105 110

Lys Leu Thr lie Met Thr Gly Asp lie Lys Gly lie Met Gin Ala Gly 115 120 125

Lys Arg Ser Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys 130 135 140

Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr 145 150 155 160

Phe Leu lie Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg 165 170 175

Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr 180 185 190

Thr Asn lie Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp 195 200 205

Ser Lys Leu Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala 210 215 220

Asp Met Gly Tyr Trp lie Glu Ser Ala Leu Asn Asp Thr Trp Lys lie 13 Ι33ύ626 " 225 230 235 240

Glu Lys Ala Ser phe He Glu Val Lys Ser Cys His Trp Pro Lys Ser 245 250 255

His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie He Pro 260 265 270

Lys Asn Phe Ala Gly Pro Val Ser Gin His As Tyr Arg Pro Gly Tyr 275 280 285

His Thr Gin Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp 290 295 300

Phe Asp Phe Cys Glu Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly 305 310 315 320

Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu lie 325 330 335

Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg 340 345 350

Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu lie Arg Pro Leu Lys Glu 355 360 365

Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala 370 375 380 14

Claims (1)

1336626 ) (4) Shadow ' | Patent No. 098133581 - Zhongli Fan p revised this November 1999 announcement Ϊ f ir -r VII, application for patent model: year 11. 11. No. 1. Used for an animal platform a pharmaceutical composition for preventing flavivirus infection, the pharmaceutical composition comprising a modified vaccinia virus Ankera strain (MVA) viral vector, the MVA viral vector carrying a performance cassette, 5 wherein the expression card S comprises a transcriptional regulation And a nucleic acid sequence encoding a complete flavivirus NS1 protein of dengue virus serotype 2 or an antigenic epitope thereof and selectively encoding another peptide/protein, and wherein the other is successful The peptide/protein is not a complete flavivirus E-protein, and 10 wherein the flavivirus infection is selected from the group consisting of dengue virus serotype 1, dengue virus serotype 3, dengue virus serotype 4, Japanese encephalitis virus , and infections caused by the West Nile virus's flavivirus. 2. The pharmaceutical composition of claim 1, wherein the composition is for treating or preventing the animal to additionally combat a derivation of the 15 nucleic acid sequence, the NS1 protein or an antigenic epitope thereof. A flavivirus infection caused by dengue virus serotype 2. 3. The pharmaceutical composition according to claim 1 or 2, wherein the flavivirus infection is caused by a mosquito-borne flavivirus. 4. The pharmaceutical composition according to claim 3, wherein the mosquito-borne yellow virus is a dengue virus. 5. The pharmaceutical composition according to claim 4, wherein the mosquito-transmitted flavivirus is a dengue virus. 6. The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid sequence encoding the NS1 protein of the flavivirus or an antigenic epitope thereof is preceded by an ATG codon and a sequence encoding a glycation signal. The sequence 'and wherein the coding sequence is terminated by a translation stop codon. 7 ♦ A pharmaceutical composition according to claim 1 or 2, wherein the transcription regulating element is a poxvirus promoter. 8. The pharmaceutical composition of claim 1 or 2, wherein the MVA viral vector is freeze-dried. 9. The pharmaceutical composition according to claim 1 or 2, wherein the composition is supplied for initial vaccination or additional vaccination, or both primary vaccination and additional vaccination. 10. The pharmaceutical composition according to claim 1 or 2, wherein the composition is formulated as a set, the set comprising the MVA viral vector, which is used for the first vaccination (first time) Inoculated) in the first vial/container and in a second vial/container for the second inoculation (additional inoculation). U. The pharmaceutical composition of claim 2, wherein the performance card encodes a protein comprising an amino acid sequence such as SEQ ID NO: 1. 12: The pharmaceutical composition of the patent application scope, wherein the MVA carrier vector is characterized in that it can replicate in chicken embryonic fibroblasts (CEF) and baby hamster kidney cell line BHK, but in human keratinocyte cell lines. There is no ability to copy in the heart. 13. The use of two types of inclusions, which are used for the treatment of "animal treatment or ribovirus" or the use of vaccines, 1336626, wherein the expression card contains a transcriptional regulatory element and a nucleic acid sequence. The nucleic acid sequence encodes a complete flavivirus NS1 protein of dengue virus serotype 2 or an antigenic epitope thereof, and selectively encodes another peptide/protein, and wherein the other peptide/protein is not 5 is a complete flavivirus E-protein, wherein the flavivirus infection is selected from the group consisting of dengue virus serotype 1, dengue virus serotype 3, dengue virus serotype 4, sputum encephalitis virus, and west Infection caused by the yellow virus of the Nile virus. 14. The use of claim 13 wherein the agent or vaccine is for treating or preventing the animal to additionally combat a derivation of the nucleic acid sequence, the NS1 protein or an antigenic epitope thereof. Flavivirus infection caused by virion 2 of leather virus. 15. For the use of claim 13 or 14, wherein the flavivirus infection is caused by a mosquito-borne flavivirus. 15 16. The use of claim 15 wherein the mosquito-borne flavivirus is a dengue virus. 17. For the use of claim 16, wherein the mosquito-borne flavivirus is a dengue virus serotype 2. 18. The use of claim 13 or 14, wherein the sequence encoding the NS1 protein of flavivirus 20 or an antigenic epitope thereof is preceded by an ATG codon and a sequence encoding a glycation signal sequence, and wherein The coding sequence is terminated by a translation stop codon. 19. The use of claim 13 or claim 14, wherein the transcriptional regulatory element is a poxvirus promoter. 3 20. The use of claim 13 or 14 wherein the mva viral vector is freeze-dried. 21. For the use of claim 13 or 14, wherein the medicament or vaccine is for use in a primary or additional vaccination, or both initial and additional. 22. The use of claim 13 or 14, wherein the medicament or vaccine is formulated as a set comprising the MVA viral vector in a supply for the first vaccination (first time) Inoculated) in the first vial/container and in a second vial/container for the second inoculation (additional inoculation). 23. The use of claim 13 or claim 14, wherein the performance card encodes a protein comprising an amino acid sequence such as SEQ ID NO: 1. 24. The use of claim 13 or 14, wherein the MVA viral vector is characterized by replication in chicken embryonic fibroblasts (CEF) and baby hamster kidney cell line BHK, but in human keratinocyte cell line HaCaT No replication capability.