TWI354560B - Flavivirus ns1 subunit vaccine - Google Patents

Description

Sixth, the invention description:

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. [Prior Art 1 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, Japanese encephalitis virus and yellow fever virus (virus field, edited by FieldsB.N' Lippincott-Raven Publishing Du, 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 encapsulated, single-strand, positive-strand RNA virus formed by three structural proteins of the dengue virus: coat protein (C) 'which forms a core 1354560 shell that binds to the viral genome, which is attached to the sputum (membrane) and E The (envelope) protein is surrounded by a fat bilayer. The genome is nearly llkb long and contains a single open reading frame architecture that envisions a multi-protein 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 followed by seven non-structural proteins: NS NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (Lindenbach and Rice, 2001) The glycoprotein NS1 found in all flaviviruses seems to be indispensable for virus viability. The dengue virus NS1 is secreted by infected mammalian cells and is in a soluble hexamer form (Flamand et al., 1999). This non-covalently bound hexamer complex is formed from three dimeric subunits and has a molecular weight of 3 l〇kDa. If it is still the only viral intrinsic protein on the surface of the infected cell, the dimerization system 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 in the form of soluble eggs (presenting a higher hexamer oligomeric form), but is secreted in combination with micro: particles rather than virions. Furthermore, it has been found that NS1 circulates in the serum of patients infected with the genovirus, suggesting that the secretion of NS1 may be an important factor in the infection of human hosts by S virus. During infection with flaviviruses, 'MSI proteins induce a strong antibody response to help clear the infectious virus in the host', which may pass through the complement regulatory pathway (Schlesinger, JJ et al., 1987) and antibody-dependent cellular cytotoxicity (ADCC). (Schlesinger, JJ et al., 1993). 4 1354560 t *

Dengue fever and its four serotypes, dengue virus serotype 1 (Den-D to dengue serotype 4 (Den-4), is very important for the flavivirus infection of humans, and the range of diseases it produces is from influenza Symptoms to severe or fatal diseases, accompanied by shock dengue hemorrhagic fever. The outbreak of dengue fever has been an important public health problem in tropical and subtropical regions with dense populations and a large number of mosquito vectors, caused by mosquito-borne flaviviruses. The spread of dengue infection and other diseases has led to more efforts in many parts of the world to develop dengue vaccines that prevent dengue fever (DF) and dengue hemorrhagic fever (dhf) and to protect individuals who have been vaccinated from Vaccines caused by some or all of the mosquito-borne flavivirus infections. Although most DF cases show signs after first infecting any of the four serotypes, most DHF cases occur in patients. Secondary sensation - the first time when different dengue viruses of different serotypes are infected. This hypothesis is generated 'gp--appropriate interval, infecting individuals with antibodies against __ dengue serotypes in order to infect different viral sera 5L, may result in a specific number of cases. Antibody-dependent increase (class) It has been proven in vitro in dengue fever and its enveloped virus, and is considered an important mechanism in the development of disease. It can also be noted that dhf IV) is in the region of viral serotype DHF. DHF cases in the older population are less likely to have multiple DHF locations (three or regions of transmission). In countries such as Southeast Asia, the incidence of specific age is higher in children, and many sero-wide The increase, which is generally consistent with the natural infection of dengue fever, can cause protective immunity. This is now 5 1354560 is different from that observed in viral infections such as hepatitis A. Clinical rank Observations show that patients may experience secondary DHF (Nimmannitya et al., 1990), but this is extremely rare and difficult to be precise. The type of serotype that leads to 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 area. Essentially, in the same individual, two or three dengue virus serotype infections may produce antibodies to the paternal reaction or even a reactive cytotoxic lymphoid reaction.

should. This may regulate or protect 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, the black spot mosquito, Egypt. 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. So, development

A fully effective vaccine 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 be to prevent infections caused by all types of ticks, so that continuous infection will not occur. The main purpose of the W〇98/13500 program is to use a recombinant modified bovine virus touch (four) that expresses all dengue fever '3L k, or four recombination, each of which has at least one The antigen of the dengue virus subtype. Both methods are compatible with all dengue fever subtypes from non-*good vaccines. However, it is desirable to introduce an early unit vaccine, which is administered 6

S can produce an immune response against more than one flavivirus or more than one dengue virus serotype, preferably against all dengue virus serotypes. 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: Embodiment 2 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 dengue virus subtype, it can be solved by providing a dengue virus = other dengue serotype 2 and a separate _ sequence of the protein or part thereof. Silk Care - The individual is at least free of all dengue subtypes: Infected 'and better for other flavivirus infections (especially mosquito-borne flaviviruses such as Japanese 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 cross-reactive antibody response. Thus, the NS1 protein from a dengue virus serotype is a universal DHF subunit for the simultaneous protection of all ash-types against dengue virus and further against other viruses of the genus Flavivirus. Because the E-protein in this unit vaccine strategy does not have the risk of antibody-dependent increase (ADE) in subsequent exposure to any serotype of dengue virus, vaccine-related DHF is transmitted in dengue fever. It should not be induced during a natural outbreak. According to a preferred embodiment, the invention relates to a DNA comprising a cassette which encodes at least a 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. In this article, s#,, 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 of the flavivirus RNA virus' Therefore, the DNA encoding the flavivirus NS1 protein is a non-naturally occurring DNA such as cDNA or synthetic DNA. The term, the expression of the flavivirus NS1* white or its singularity is "cutting the virus NS1 protein or part thereof" The coding sequence "Essence (especially the origin of transcription). About this transcript nuclear activator / enhancer. Difficult eukaryotic _ sub / 増 strong sub-system human giant ^ cell virus immediate early promoter / enhancer and as The example part of the road 'such as the U starter, the epidemic virus minimum promoter of the epidemic virus promoter." The most viral mobilization of the epidemic virus solution in Figure 2 and for the money: 9., if necessary, the performance Card E may further include elements that control the termination of transcription, such as pronuclear termination elements or eukaryotic multiple A signal sequences. = The performance card E may only represent NS1 * white of the flavivirus or its part or may be expressed together with - or more An additional yellow virus protein/peptide protein or its fraction, The protein or portion thereof and the additional protein/Shengyue Tai are formed into a protein/peptide or a fusion protein/peptide form. The term "peptide" in the context of the present invention is not defined herein. Means that at least one amino acid is longer than 20 amino acids, more preferably a continuous amino acid sequence of 25 amino acids. The additional flavivirus protein is not a complete £_protein because of this protein It seems to involve the development of DHF. Therefore, if the additional flavivirus peptide is born from E-protein, it should contain less than 40 amino acids, preferably less than amino acids. If derived from E - the long sequence of the amino acid sequence of the protein is expressed together with the NS1 protein or a portion thereof, and it should be confirmed that the amino acid long column does not contain an epitope involved in the production of ADE # DHF. In addition to the NS1 protein or a portion thereof, The performance cardinal also exhibits an additional flavonoid/sheng peptide in the form of an octaprotein/span form, and the performance is between the sequence encoding the MSI protein or a portion thereof and encoding additional flavivirus protein < May contain a ribosome in place (IRES) IRES elements are known to those skilled in the art. Examples of IRES elements are the small RNA virus IRES element or the 5' uncoded region of hepatitis C virus. Alternatively, the nuclear acid sequence encoding the NS1 protein or a portion thereof Can be fused to a DNA sequence encoding an additional flavivirus protein/peptide, whereby a fusion protein is formed between the NS1 protein or a portion thereof and the additional flavivirus protein/peptide. If the NS1 protein or In part, the portion of the flavivirus protein/Shengyuetai is to be formed into a fusion protein/peptide, and the respective coding sequences are fused in frame. In a preferred embodiment A sequence encoding a glycated signal sequence encoding an E-protein is added before the DNA sequence encoding the NS1 protein or a 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 E-protein-derived amino acid long column should be as short as possible and it should be excluded from the long list of amino acids that would contain epitopes involved in Ade and DHF production. The E-protein glycation signal sequence meets this requirement. In a preferred embodiment of the invention, 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 card E of the present invention does not exhibit any other peptide/protein from other flavivirus genome portions, particularly NS2A or E proteins. In another optional embodiment, the DNA of the present invention exhibits an NS1 protein or a portion thereof which is a fusion protein form protein having a protein/peptide which is not derived from flavivirus/Shengyuetai contains a non-yellow virus signal sequence. Or for the extraction or purification of a sequence of a expressed fusion protein such as a tag. 1354560 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 followed by the host cell protease Interrupted and then disrupted 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 glycosylated 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 3, terminal TAG of the protein added to 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 squirrels (,, NGC cultivar), genetic data The library accession number AF038403) starts with 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: 1. Therefore, in short, this A specific example relates to a DNA comprising a cassette which exhibits a sequence encoding a flavivirus NS1 protein or a portion thereof, wherein the coding sequence is preceded by a start codon ("ATG") and - The sequence encoding the signal sequence for saccharification is preferably derived from an E protein as defined above and wherein the coding sequence is finally translated with a stop codon (Figs. 1A, 1C and 2, SEQ ID NO: 5-10). The DNA sequence of the present invention encodes a flavivirus NS1 virus or a portion thereof. The term "flavivirus" means any flavivirus. More preferably, the term "flavonoid" means, for example, 11 1354560 West Nile virus, Japanese encephalitis virus Yellow fever virus and A mosquito-borne flavivirus of dengue virus. A NS1 protein or a portion thereof derived from a mosquito-encoded mosquito encoded by the DNA of the present invention should protect the vaccinated individual from infection of the virus or virus subtype derived from the vaccine, and is exempt from the Other infections of mosquito-borne viruses or other viral subtypes of the vaccine. The NS1 protein may preferably be any dengue virus subtype. More preferably, the NS1 protein coding sequence is derived from a dengue subtype 2 such as the dengue virus neonatal squirrel breed (NGC variety, Gen. No. AF038403). The terms "subtype," and "serum type" are This invention is used interchangeably. The term "NS1 protein, or a portion thereof", means a long column of amino acids of the NS1 protein, which is long enough to induce a specific immune response against the NS1 protein derived from the "partial" portion thereof. . If the flavivirus is a dengue virus, the long list of amino acids should be a long list of amino acids that induce an immune response against the nsi protein of all dengue virus subtypes in vaccinated animals, including humans. It is shown in the Examples section how a person familiar with this technique can determine whether the NS1 protein or part thereof induces a specific immune response to all of the Dengya subtypes. According to a preferred embodiment, the flavivirus sequence encodes the entire NS1 protein.

Briefly, one of the best embodiments of the present invention is a mosquito containing a bat code.

The spread of the flavivirus NS1 or a part thereof of the sub-transfected flavivirus is preferably dengue, more preferably dengue virus subtype 2, and the expression of the white or part thereof is controlled by eukaryotic transcription regulating elements. Wherein the yellow disease NS1 egg is more preferably the DNA of the present invention encodes an NS1 protein or a portion thereof in the form of a fusion protein having a glycosidation signal sequence. / The present invention further refers to a vector comprising the DNA of the present invention.谇% 12 1354560 • The term "carrier" means any person known to the art. The carrier may be a plastid vector such as PBR322 or a vector of the pUC series. More 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 to be selected into the viral genome of the respective viral vector. The recombinant viral genome is then packaged so that the resulting recombinant vector can be used to infect cells and cell lines, particularly living organisms including human animals. A typical viral vector according to the invention is an adenoviral vector, an enterovirus vector or a vector based on adeno-associated virus 2 (AAV2). The best is the carrier of poxvirus. Preferably, the poxvirus is a canary virus, a poultry cancer virus or a bovine cancer virus. More preferred is the modified vaccinia virus Ankara (MVA) (Sutter, G et al, [1994], Vaccine 12·1032-40). A typical MVA variety is MVA 575, which is deposited at the European Animal Cell Culture Collection Center under the registration number ECACC V00120707. More preferably, MVA-BN or a derivative thereof, as described on November 22, 2001, is modified by the Bamarian Nordic Research InsUtute GmbH. The vaccinia Ankara virus variant "is titled in the PCT application filed by the European Patent Office. MVA-BN has been deposited in the European Animal Cell Culture Collection Center under the accession number Ecacc V00083008. By using MVA-BN or Derivatives, additional technical problems have been solved, providing a particularly safe viral vaccine against flaviviruses, as it has been shown that the MVA-BN viral vector is a completely attenuated virus derived from the modified vaccinia Ankara virus and It is characterized by loss of its ability to replicate efficiently in human cell lines. MVA BN is safer than any other known bovine cancer virus species due to the lack of replication capacity in humans. In a preferred embodiment,

C 13 1354560

The invention relates to a kind of cryptic containing the _, and the mva bn derivative carrier MVA BN, allowing evaluation of whether it is a sputum, or a manalysis of a derivative of MVA BN (4) (4) and allowing obtaining mva bn or a derivative thereof The method is shown in the following description section and in the third embodiment. The term "derivative" of the virus, such as the "derivative" of the virus of the registry number ECACC v〇〇〇83_ (i.e., a derivative of MVA.), means at least one frequency of the hosted species is indicated. But in its base _ or multiple parts show the difference between the vaccinia virus. Preferably, the derivative has at least two, more preferably all of the features of the derivative having at least three, more preferably the following four MVA_BN. In particular, the derivative of MVA BN has substantially the same identity as jjVA-BN Copy features. The same replication characteristics as the deposited virus, the virus means that in the CEF cells and cell lines BHK 'HeLa, HaCat and 143B, the virus has a similar amplification ratio to the registered variety' and when the AGR129 gene is transferred When the mouse model is detected, it shows a similar copy in vivo. The characteristics of MVA-BN are as follows:

- Can replicate efficiently in chicken embryonic fibroblasts (CEF) and baby hamster kidney cell line BHK (ECACC 85011433), but not in human cell line HaCat (Boukamp et al., 1988, J Cell Biol 106 (3) ): 76b 71), - unable to replicate in vivo, - induces higher immunogenicity in the mutual stimulation model compared to the conventional MVA575 (ECACC V00120707) and / or - when compared to DNA - initial / Vaccinia virus push-up therapy induces at least substantially the same level of immunity in vaccinia virus primary/vaccinia virus push-up therapy. The term ''ability of no effective replication" means that the virus of the present invention exhibits a replication rate of less than 1 in the human cell strain HaCat (Boukamp et al., 1988, J Cell 14 1354560 •

Biol. 106(3): 761-71. Preferably, the virus used as the vector of the present invention has an amplification ratio of 〇.8 or less in the human cell strain HaCat. The "amplification ratio of a virus, the ratio of the virus produced from an infected cell (output) relative to the number (input) used to infect the cell at the first location ("amplification ratio"). The ratio of the output to the input of "1" is defined as an amplification state in which the number of viruses generated from the infected cells is the same as the number originally used to infect the cells. This state implies a fact that is infected The cells allow viral infection and viral replication. The optimal MVA vector, particularly MVA-BN and its derivatives, does not replicate efficiently in HaCat• cells. In particular, MVA-BN is in human embryonic kidney cell line 293 ( The ratio of the amplification ratio shown in ECACC No. 85120602) is from 0.05 to 0.2. In the human osteosarcoma cell line 1436 (£0 pills 0: sen. 91112502), the ratio of the ratio is 0.0 to 0.6. Within the scope of human 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), etc. The range of the amplification is in the range of 0.04 to 0.8 and 0.02 to 0.8. The amplification ratio of VA-BN in African green monkey kidney cells (CV1: ATCC No. CCL-70) is 0.01 to • 〇·06. Therefore, MVA-BN is not effective in any human cell strain tested. Copy. The amplification ratio of MVA-BN in chicken embryonic fibroblasts (CEF: primary culture) or baby hamster kidney cell line BHK (ATCC No. CRL-1632) is clearly greater than 1. As outlined above, greater than "1" The ratio means effective 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 easily be found in CEF at a ratio greater than 5 〇〇. In culture, and multiply and expand in BHK cells at a ratio greater than 50. 15 In the context of defining MVA-BN, the term "cannot be replicated in vivo, means that the virus cannot be human and mouse models as described below. Copy in . This "cannot be replicated in vivo, preferably in mice that do not produce mature B and T cells". An example of such a mouse is the genetically transgenic mouse model AGR129 (available from Mark Sutter, Institute of Virology, University of Zurich, Switzerland) This mouse cultivar has a genetically-labeled glycosidic interference in the IFN receptor type I (IFN-α/β) and type Π (IFN1) genes and RAG. Because of this glucoside interference, the mouse does not have a 1FN system and cannot produce maturation. B and T cells, and for themselves, are severely immune to abandonment and highly susceptible to replicating viruses. Except for AGR129 mice, any other immune abandonment and height that are unable to produce B and T cells and are severe in their own right A mouse species susceptible to the replication virus can be used. In particular, the virus of the present invention is preferably at least 45 days after infection with AGR129 mice by intraperitoneal injection of 107 pfu of virus, preferably 60 days, preferably. The mouse is not poisoned for a period of 90 days. Preferably, the virus showing "unable to replicate in vivo" is further characterized by infection of AGR129 mice by intraperitoneal injection of 107 pfu of virus. The next 45 days 'better 60 days, the best is 90 days, no virus can be recovered in the organs or tissues of the mouse. According to the results of the lethal stimulating mouse model, the characteristics of MVA-BN and its derivatives are better. It is more immunogenic than the known MVA 575. In this model, 'no vaccinated mice are in Western reserves (Western

Reserve) Variety L929 TK+ or IHD-J can fully replicate the vaccinia variety and die after infection. 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 in the standard spot analysis by using VERO cells. The virulence titer of the disease was measured in the ovary. The virus titer in the mice which were not vaccinated and the mice inoculated with the vaccination virus according to the present invention was determined. More specifically, the virus of the present invention is characterized by In this test, the virus titer in the ovary after inoculation with 102 TCID5D/ml as the virus of the present invention was reduced by at least 70% compared to the non-vaccinated mouse, preferably 1% less than 8%, more preferably at least 90%. %. MVA-BN or a derivative thereof is preferably characterized by at least substantially inducing vaccinia virus primary/vaccinia virus ascending therapy compared to DNA-primary/vaccinia V disease. The same immunological level. Compared with the DNA-primary/vaccinia sputum push-up therapy, if the vaccinia virus primary/vaccinia virus push-up therapy is used in the following two analytical methods (,, analytical method 1 ", and, analysis Method 2"), preferably When the CTL responses measured in parallel are at least substantially the same, then the vaccinia virus is considered to be boosted by the vaccinia virus primary/vaccine virus compared to the DNA-primary/vaccinia virus push-up therapy. At least substantially the same immunological level can be induced. More preferably, compared to the DNA-primary/vaccinia virus push-up therapy, measured by at least one assay after administration of the vaccinia mother's primary/vaccinia virus boost. The CTL φ reaction system is more preferred. The best one is that the CTL reaction is more than the following two methods: the vaccinia virus initial/vaccinia virus push-up administration method, by intra-abdominal/main shot 1〇7 TCID5. As shown in the present invention, a murine polyhedron vaccinia virus (described in Th〇mson et al., 1988, J. Immunol. 160, 1717) is used for the first time - immunization of 8 weeks old BALB/c (H- 2d) Rats, and after three weeks, the same number of viruses were administered in the same manner to boost-immunize the mouse. For this purpose, it is necessary to construct a recombinant bovine virus system that expresses the polyhedron. Constructing the recombinant vaccinia The method is known to those skilled in the art, and In the text, in detail, when the DNA is firstly/vaccinia virus push-up therapy, the initial inoculation is carried out by intramuscular injection of the mouse 50 Mg at 17 1354560, which will be expressed in the same way as the DNA of the same antigen as the vaccinia virus; / Vaccinia virus pushes the same method to carry out the promotion of vaccinia virus. In the publication of Thomson et al. cited above, the DNA plastids that express polyhedra are also described. The development of CTL responses against the epitopes SYIPSAEKI, RPQASGVYM and/or YPHFMPTNL was determined two weeks after the boost administration. The determination of the CTL reaction is preferably carried out using ELISP0T analysis (described in

Schneider et al., 1998, Nat. Med. 4, 397-402), and a specific virus according to the present invention is outlined in the Examples section below. The characteristics of the virus according to the present invention are in this experiment, when evaluated by the number of cells producing IFN_y/1 〇 6 spleen cells, and the vaccinia virus priming/vaccinia virus is pushed up and attacked, against the upper side. The CTL immune response of the epitopes mentioned 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 the analytical method. However, instead of intravenously administering 1〇7 TCID5 as in Analytical Method 1. Vaccinia virus, in this assay, was administered subcutaneously with 108 TCID5. The vaccinia virus of the present invention is used for primary immunization and boosting immunization. The characteristics of the virus according to the present invention are evaluated in this experiment when the number of cells producing IFN-γ/1〇6 spleen cells is evaluated against the above-mentioned vaccinia virus priming/vaccinia virus boosting administration. 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). It is well known to those skilled in the art how to obtain the properties of ΜνΑ-βΝ and its derivatives as shown above: The method for obtaining this virus may comprise the following steps: 18 1354560 * t - introduction of a known vaccinia virus The cultivar 'preferably MVA 574 or MVA 575 (ECACC V00120707) is for a non-human cell in which the virus can be efficiently replicated, wherein the non-human cell is preferably selected from CEF cells and cell line BHK, - isolated/rich from The virions of the cells, and - whether the virus obtained by the assay has at least one biological property as desired above,

The above steps can optionally be repeated until a characteristic virus is obtained. Having the desired replication method for introducing a recombinant poxvirus into a poxvirus DNA, such as the DNA of the present invention, is well known to those skilled in the art. In the poxvirus, the performance of the DNA of the present invention is preferably under the transcriptional control of the vaccinia virus promoter, but it is not absolutely clear that the DNA is preferably a non-essential region inserted into the viral genome. & In another preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at a position where deletion occurs naturally in the MVa gene line (disclosed in PCT/EP96/02926).

The law as well as the ~ recombination of cattle, more. As a preferred embodiment of the present invention, a vector comprising the DNA of the present invention, wherein the vector is MVA_BN or a derivative thereof, and wherein the DNA of the present invention comprises a dengue virus encoding In particular, the performance of the NS1 protein of the dengue virus blood type 2 or a part 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 < DNA or a vector according to the invention. For the definition of NS1 or a portion thereof of this specification, reference is made to the above section of the specification, wherein the invention is defined by the product of the DNA representation. Therefore, the following related as this 19 1354560

A brief description of the white matter is not to be construed as limiting the invention. Briefly, the protein as in this month may be a separate NS1 protein or portion thereof encoded by any flavivirus. Preferably, the NS1 protein or portion thereof is derived from a dengue virus subtype 2 derived from a dengue virus. The protein of the invention may only be the amino acid sequence of the S NS1 protein. In a preferred embodiment, the NS1 protein may comprise an additional amino acid which is essential for the efficient expression of the protein f. Examples of such amino acid/amino acid sequences are shown above and include at the N-terminus of the protein a methionine encoded by the ATG codon to which it is added, and - from the E-protein. The amino acid sequence functions as an egg material for saccharification of the present invention. Other signal sequences are also included in the scope of the present invention. In the optional embodiment, the cis-amino acid sequence or a portion thereof can be fused to other proteins/success. Examples of fused partners are sequences that allow for the identification of proteins, such as tags or other flavivirus proteins or portions thereof. In the preferred embodiment, the present invention relates to the present invention as a vaccine.

Ship, carrier or (iv) protein or part thereof. , the vaccine "is a compound that induces a specific reaction of DNA, protein 'vector or virus. According to the optional example, the invention, the vaccine "system, according to - dengue virus prion protein or its - part It induces an immune response against deproteinization of all dengue virus subtypes. In particular, it has been shown that the dengue virus subtype of the curry two == anti-sputum produces an immune response against all dengue virus subtypes of the NS1 protein 'better against other mosquito-transmitting hosts: as mentioned above, this The inventor of the invention discovered that the virus of the NS1 20

1354560 • I

The protein or part thereof induces an immune response against the NS1 protein of other flaviviruses. As indicated above, "a flavivirus, preferably a mosquito-borne flavivirus. In other words, the (4) human of the present invention finds, in the specific case of the present invention, such as the NS1 of the mosquito-borne flavivirus of the present invention. The protein or part thereof induces an NS1 protein that is resistant to the mosquito-borne flavivirus derived from the vaccine and an immune response against other mosquito-borne flaviviruses. Therefore, it is derived from the mosquito-borne κ disease The field may be used as a carrier against the yellow virus of a plurality of mosquito-borne flaviviruses, or a similar term herein means a carrier of the invention (such as an epidemic viral vector or plastid). ). Therefore, the imaginary body inlay is not the carrier skeleton. "A vector derived from a flavivirus," an example of which is a virulence vector such as MVA, which comprises - a performance card g, a calcivirus promoter, a sequence encoding a flavivirus NS1 protein or a portion thereof, wherein the coding The sequence of the flavivirus NS1 protein or a portion thereof is preceded by a sequence of a TM codon and a - bat saccharification signal sequence, and wherein the terminal of the transcript sequence is a translation-terminated terminator.

Thus, a vector or a protein-like vaccine having the present invention can be used as a single-volume vaccine against I extensive yellow flavivirus or at least a flavivirus subtype. The vaccine derived from the sputum virus subtype 2 can be derived from the (four), the nsi ^« part of the dirty ' or the Ns 1 protein from the flavivirus or subtype or a part thereof can be used as the electric (IV) As a vaccine against subtypes 1, 3 and 4 and against subtype 2. Complex and other flavivirus protection against virulence - the individual is against a disease such as West Nile. In a preferred embodiment, the brain of the present invention is vaccinated. Administration of naked DNA containing 21 1354560 of the eukaryotic expression cassette of the present invention, particularly intramuscular injection of DNA, can result in the expression of the protein encoded by the expression cassette, as is known to those skilled in the art. . The protein is exposed to the immune system and produces a specific immune response. In a preferred embodiment, the vaccination of the vaccine is carried out by administering a vector of the present invention, particularly a viral vector, more preferably a poxvirus vector, preferably a vaccinia virus vector such as an MVA vector. In the preparation of a vaccinia virus-based vaccine, the virus of the present invention is converted into a physiologically acceptable form. This preparation is based on the experience in preparing a cancerous field against smallpox. (described in Stickl, H. enriching, [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, it will be pure

The virus is stored at -80. 〇 was formulated at a titer of 5xl08TCID5e/ml in about 10 mM Tris, 140 mM NaCl pH 7.4. In the preparation of vaccines, '1〇2-108 virions are lyophilized in ampoules in 10〇ml, phosphate buffered saline (pBS) with 2% protein gland and 1% human albumin. 'It is better to be a glass ampoule. Optionally, the vaccine can be produced by gradual freeze-drying of the virus in the formulation. The formulation may comprise additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or such as antioxidants or inert gases, stabilizers or proteins suitable for in vivo administration (eg human serum albumin) Other additives. The glass ampoule is then sealed and it can be stored at 4 ° C and room temperature for several months. However, as long as it is not necessary, the ampoule is better stored at temperatures below -20 °C. When inoculated, the lyophilizate can be dissolved in a liquid solution of 〇·1 to 0.5 ml, preferably physiological saline or Tris buffer night, by systemic or local administration, that is, by parenteral or intramuscular. Or other investment methods known to those involved in the work of 22 people. The number of modes of administration, dosage, and administration can be optimized by those skilled in the art. The optimal mode of administration of poxvirus vectors is subcutaneous or intramuscular administration. 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 a vaccine kit comprising the MVA-DN viral vector of the present invention, which is for use in a first vaccination ("first") in the first vial/container and a second vaccination in the second vial/container (", push up"). If the vaccine is a DNA comprising the present invention The MVA-BN vector or its neoplasm, a specific specific example of the present invention relates to the administration of a therapeutically effective treatment at the time of the first vaccination ("primary vaccination") and the second vaccination ("push vaccination"). The vaccine according to the invention is therefore in the case of a vaccine, the invention relates to a vaccine comprising the MA, the vector or the NS1 protein of the invention or a part thereof, and the use of the DNA, vaccine or protein for the preparation 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 subtype, and 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 is more related to the treatment or prevention of flavivirus infection. A method comprising, in an animal comprising a human being in need of treatment or prevention of a flavivirus infection, inoculation of a DNA as described above, a vector as described above or an NS1 protein as described above or a portion thereof. In particular, the present invention relates to a A method 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, and wherein the DNA, the vector or the NS1 protein or a portion thereof is used as a counteracting Vaccine of dengue subtype. Brief Description of the Invention A DNA comprising a performance cassette encoding at least one flavivirus NS1 protein or a portion thereof, such as the DNA described above, wherein the expression cassette comprises eukaryotic transcriptional regulatory elements And a sequence encoding a flavivirus NS1 protein or a portion thereof, such as the DNA described above, wherein the NS1 protein encoding a flavivirus or a portion thereof The sequence is preceded by an ATG codon and a sequence encoding a glycosylated signal sequence, and wherein the coding sequence ends with a translation stop codon. The DNA as described above, wherein the eukaryotic transcriptional regulatory element is a poxvirus The DNA of the above, wherein the flavivirus is a mosquito-borne flavivirus, such as the DNA described above, wherein the flavivirus is a dengue virus, such as the DNA described above, wherein the dengue virus is a dengue virus subtype 2. A vector comprising the DNA as described above, wherein the vector is a poxvirus vector, such as the vector described above, wherein the marker is a bovine virus, particularly a modified bovine cancer 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 seedling of 1354560. 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 or a portion thereof encoded by the DNA or the vector 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 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 for the transmission of a vaccine (i.e., 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 brain as described above, a vector such as the above, an NS1 protein as described above or a part thereof to an animal in need of treatment or prevention of the flavivirus infection, including humans . The method as described above, wherein the NS1 protein or a portion thereof or the NS1 protein or a portion thereof encoded by the vector or the vector is derived from a dengue virus subtype, wherein the brain, the vector, the (4) S1 protein or a portion thereof It is used as a vaccine against all dengue virus subtypes.

Use as described above, wherein the NS1 protein or a portion thereof or the NS1 protein encoded by the viscera or the vector or a portion thereof is derived from a mosquito-borne flavivirus, wherein the DNA, the vector or the NS1 protein or Partially used as a mosquito-borne flavivirus against a derivative vaccine (ie, the DMA 1 vector or the material or its material) and against other mosquito-borne flaviviruses. Example

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 technology provided by the invention to such embodiments. Example 1: Composition of mBN07 1. Detailed description of NS1 antigen (Fig. 1) This example relates to NS1 of serotype 2 from the Scorpio mouse C-NGC variety (e.g., gene library sequence AF038403). Since the NS1 protein of the flavivirus is produced as part of the polyprotein precursor, the NS1 gene is not preceded by the ATG "start codon" in terms of DMA. Therefore, the cDNA sequence encoding the NS1 protein must be added. One "ATG" Start 26 1354560 • Codon. This is followed by a signal sequence whereby the newly synthesized NS1 protein is glycosylated in the endoplasmic reticulum. Finally, the protein-encoding cassette requires a termination. 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 a hydrophobic C-terminal derived from E protein. (At least 28 amino acids, for NGC varieties. Starting with amino acid M (ATG). ' Figure 1A shows the exact signal sequence + NS1 φ sequence as an embodiment of the invention (see also SEQ ID NO: 5 and 6). The "signal sequence + NS1" nucleotide coding sequence was obtained by RT-PCR amplification of the dengue NGC RNA genome by using the following primers: D2NS Bu 1 above: 5' -ACAdM7T7IiGAAI £AATTCACGTAGCACCTCA-3' (SEQ ID NO: 4) Italicized part: Bgl II endostriction restriction enzyme recognition position. Underlined is the start codon. φ D2NS 2 2 below: 5, -AATJM7rTCTA£IAGGCTGTGACCAAGGAGTT-3' (sequence s horn 3) ^ Italicized part · Bgl II endonuclease restriction enzyme recognition position. Underlined is the stop codon. According to the manufacturer's recommended instrument 'This RT_pCR amplification can be used from

Roche Molecular Biochemical's Titan 〇ne playing, coffee group (catalog number 1-939-823). However, essentially any commercially available set of uncommercial RT-PCR kits can be used instead. After North* or 27 1354560, the RT-PCR product can be colonized into the BamH丨 position of any of the most selected colonies in many commercially available bacterial selections, however, in this example , the line was selected into ρΑΠ to be selected into pAF7D2NSl-

See Figures 1B and 1C for the detailed sequence of pAF7D2NS1. The id diagram shows the MSI 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. A short N-terminal hydrophobic region refers to a signal sequence. 2. Details of the NS1 performance cassette (Figure 2) is the ''signal sequence + NS1" from the poxvirus vector such as canarypox, poultry pox, vaccinia or MVA, on the 5' end of its cDNA Need to add, produce a virus promoter. A multi-adenosine signal sequence is not required when all of the poxvirus-synthesized RNAs are multi-adenosed with virally encoded enzymes that do not require the polyA additional sequence to perform this function. Any pox virus promoter can be used to express this cassette. Fig. 2 and SEQ ID NOs: 9 and 10 show the nucleotide sequence of the 11 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 + NS1" is further derived from the NS1 plastid clone by PCR using primers OBN338 and OBN345. The oBN345 primer contains the nucleotide sequence of the minimal promoter element 5' of the cancer virus to the target sequence within the selected plastid. The sequence of the plastoquinone that binds to the BN345 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 1354560 * * No. 1) oBN345: 5'-caaaaaattgaaattttattttttttttttggaatata^ataaaa ACACGATAATACCATGG -3' (sequence number 2) underlined The nuclear sinus acid represents the smallest promoter sequence of the pox virus. The first five cycles of the binding temperature for the PCR amplification reaction were calculated by binding the OBN345 to the nucleotide sequence of the same sequence in the selection vector. 3. Integrate the NS1 performance cassette into the MVA (Fig. 3). The PCR amplified product was selected as the terminal Xho I position (see Figure 3a) with a flat blunt end selected into the plaque of the plastid pbnx〇7 (see Figure 3a). Body pbn41 (see Figure 3a). pBN41 was used to integrate the 11 acne promoter + signal sequence + NS1" cassette into the vector of deletion position 2 of MVA by homologous recombination. The basic characteristics of pBN41 (see Fig. 3a) are as follows: The plastid skeleton is derived from. Stratagene's pBluescript SK-plus (Genbank VB0078) -D2F1: deletion of the 2 flanking 1 homologous recombination arm. This represents the nuclear sinus acid sequence from 20117 to 20617 of the MVA gene library sequence U94848. -PPr: Poxvirus promoter. -NPT 11: Neomycin disruption enzyme protein coding 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 fluorescent protein coding sequence (protein coding sequence of gene library sequence U57609 - nucleotide 675 to nucleotide 1394) » 29 1354560 -NS1: "Signal sequence + from dengue NGC variety" NS1" protein coding sequence. -D2F2: deletion 2 flank 2 homologous recombination arm. This represents the nuclear sinus acid sequence from 20719 to 21343 of the MVA gene library sequence U94848.

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 within the MVA genome

G. NS1 showed a sputum and a reporter card (pox promoter + NPT II-IRES-EGFP) was integrated into the MVA genome. This is achieved by transfecting the linear integration vector into chicken embryonic fibroblasts (CEF) which have been infected with low amounts of Mva (MOI, e.g., per cell 〇.1 infection unit). The virus extract was prepared 48 hours after infection or when the infection had reached confluence and stored at -20. (: The choice of recombinant MVA (rMVA) for selection and purification in the preparation. 3.1.2 Selection of rMVA and purification of selected plants Non-recombinant MVA exclusion (empty vector virus) and amplification of rMVA This is achieved by infecting fusion chicken embryonic fibroblast (CEF) cells in the presence of G418 (the number of G4 8 must be optimized to the highest dose that will not kill CEF cells). The virus with the integrated π gene will not replicate in the presence of G418 added to the cytoplasmic culture medium, and will inhibit the brain replication', but because the CEF cells will be in a non-replicating state, they will 30 1354560 • · Not affected by G418. Cef cells infected with rMVAs can be observed under the Dengguang microscope due to the enhanced expression of fluorescent green proteins. The virus extract from this homologous recombination step must be sequential Diluted and used to infect untreated cef cells in the presence of G418 and covered with low melting acacia gum. After 2 days of infection, the infected acacia seeds were placed under a fluorescent 'microscope Single green of infected cells Xi imaging. The Flag and the like and to remove containing • from the infected cells into a clear image of the agar gel plug, and placed in a sterile cell maintenance-containing culture solution of 1511) in a microcentrifuge tube. The virus was released from the gelatin plug by soaking the tube 3 times at -2〇〇c. The best selected strains or the selected strains were further purified by pCR analysis under acacia until no signs of contamination with the blank (3 to 30 rounds of selection). The selected strains are then amplified for further stringency testing - alignment of the inserted configuration, sequence identification of the foreign promoter gene cassette, and performance analysis by RT-PCR. After these analyses, only one of the selected strains was further expanded under the selection of G418, and a major stock solution was prepared for further study of the characteristics and immunogenicity. The recombinant MVA having the dengue performance cassette described in the present invention is called mBN07. Figure 3b shows the configuration of the foreign sequence inserted in mBN07. 4. Real NS1 by MVA The expression of the NS1 protein from this recombinant MVA (mBN07) was confirmed by standard Western blotting analysis without denaturation. Figure 4 shows the results of NS1 expression after purified mBN07 was used to infect mammalian tissue culture cells (e.g., BHK-21 cells, 1.00Ι 1.0 cells per cell). The protein extract of Rough 31 1354560 was prepared from the infected cells 24 to 30 hours after infection. 'At this time, some extracts were contained with 2-mercaptoethanol (2-ME) or 2-ME-free. The SDS-PAGE gel loading buffer was mixed. These samples, which are positive control groups, plus protein extracts from cells infected with the dengue NGC variety (mosquito cell line) were separated by electrophoresis in an SDS-PAGE gel, and then spotted on nitrocellulose. On the membrane. The membrane was probed with a monoclonal antibody against dengue NS1. Figure 4 shows that the NS1 line expressed by mBN07 is recognized by anti-dengue NS1 monoclonal antibody and forms the correct dimeric pattern similar to NS1 from dengue-infected cells (compared to no boiled '2-free ME's mBN07 Lane and Lu are not boiled, not in 2-ME DEN2 Lane). Figure 4 shows that the dimeric form is divided into monomeric forms in the case of denaturation (see the boiled, 2-ME mBN07 lane). NS1 expressed in cells infected with mBN07 can also be identified in Western blotting analysis by serum from patients with combined recovery and individual antibodies with interaction with all four serotypes of dengue fever. . This demonstrates that the NS1 line expressed by mBN07 produces immunogenicity. Example 2: NS1 expressed by mBN07 for non-serum type 2 dengue disease and toxicity and immunogenicity of NS1 of Japanese encephalitis 1. Serum reactivity test of NS1 expressed by mBN07 in reconstituted patients The results have the following possibility that NS1 expressed by mBN07 can be confirmed by the sera recovered from dengue virus-infected individuals. 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 Prepared as 32 1354560 « · Immune dot strip with cell extract as MVA-GFP infected as control group. The antigen-containing cells were treated with sample buffer containing no decylhexanol and were not heated. Serum for the test of 68 individuals, (91 2%) in the circle of immune dots reacted with BN07 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 mBN07. 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) true JEV NS1 positive 71.43% (5/7) 28.57% (2/7) Comparison of antiserum to true NS1 response to NS1 expressed by mBN07. In brackets: The number of samples tested positive / the total number of test samples. DEN = dengue, JEV = sputum encephalitis virus. The same 68 sera were also analyzed for their reactivity to the protein of the premembrane. 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 JEV anterior membrane and vice versa. 33 Analysis along these lines will provide a better forecast of the individual's infection history. Table 2 shows that serum from 22 individuals reacted alone with the true dengue pro-membrane protein' so it is speculated that the 22 patients 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 infected with both dengue and JEV, and all 22 sera also responded to NS1 by mBN〇7. 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, 17 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: True DEN prM positive BN07 NS1 negative BN07 NS1 positive 〇% (0/22) 100% (22/22) True DEN & JEV prM—positive 〇% (0/22) 100% (22/22) Real JEV prM positive 19.0% (5/7) 81.0% (17/21) PrM negative 66. 7°/〇 (2/3) 33.3% (1/3) Antiserum to true anterior membrane with „^|\1 Comparison of the responses of 〇7!^1. In brackets: The number of samples with positive s test / the total number of test samples. DEN = dengue, JEV = sputum encephalitis virus. The data in Table 2 also clearly shows that For 6 sera that did not respond to NS1 in mBN07, 4 were from individuals who had previously been infected with JEV, not dengue. The remaining 2 were not detected for either dengue or JEV 1354560 • before Membrane protein antibody may be too low in potency. 2. κιΒΝ07 inoculated rabbit and tested immunized serum against dengue virus and Japanese encephalitis virus, immuno dot map and EL ISA analysis according to the inoculation procedure of the present invention as shown below, Three rabbits without specific pathogens were immunized subcutaneously. On the third day, a bottle of sterile water was used to prepare 丨ml• of the frozen-dried Miao (Ixl0e8 TCID50 BN07 freeze-dried vaccine). Inoculate each donkey, then inoculate again on the 28th day. Take the blood sample before the first inoculation (pre-collection), and in the second inoculation 1 Take one more time after the day. Day 3 = pre-blood collection, then the first vaccination on the 28th day = the second vaccination on the 38th day = blood sampling, back to the blood collection and the side, the blood of the prayer Figure 5 shows a 1:200 dilution of serum tested in a non-denaturing condition, the immunological dot plot of the dengue 2 virus antigen isolated by SDS PAGE Control strips with uninfected G6/36 cells. The results clearly show that 'when inoculated with mBN07' all rabbits produced relatively high titer anti-de-antibody against tissues infected with dengue serotype 2 The true NS1 produced by the culture of mosquito cells produces a cross-reaction. The serum obtained at the time of inoculation does not react with any dengue protein on the immune dot map.

The immunized serum was titrated into 丨: 1〇〇〇, 1:2〇〇〇1:4〇〇〇, 丨:1〇1.10, 1.10, and was tested in the absence-denatility case by SDS PAGE 35 1354560 Immune dot strips of dengue 2 virus antigen isolated and control strips of cells not subjected to C6/36. See Figure 6 for the titration of three rabbits. The titration is calculated to be 1:10000. The serum before and after immunization is titrated to 1:10-1 2, 1:10-3, 1:10-4, 1:10, 1:10 6, 1:10·5 ' and tested in indirect IgG ELISA. The well was coated with 1:250 diluted dengue 2 and uninfected C6/36 cell lysate. Table 3 1:1 (T2 1:103 1:104 1 · 1 n-6 7 1:10 -〇. 012 0. 006 0. 007 0.623 0.127 0.02 0.004 0.001 -〇〇03 -0.012 〇-0.001 -0.003 〇 -〇'〇〇1 0.402 0.06 -0.007 0.008 -0.003 〇'〇〇2 -0.008 0.03 -0.002 0.005 -0.002 -〇〇〇〇0^907 0.224 0,038 0.011 -〇. QQi 〇〇3 Bunny #1 Bunny #2 Rabbit #3

Pre Post Pre Post Pre Post 1:10'

0.003 -0.002 〇〇T 36 1 ELISA absorbance reading of pre-immunization and post-immunization graying of each rabbit at different dilutions (pre = pre-immune serum, p〇st = post-immune serum). The titration results after immunization of each rabbit are shown in Fig. 7. The endpoint titer evaluated by Qiqing for each rabbit immunization was 1:1〇〇〇. 2 2 pre-operative blood and acid-free serum hang dengue fever hi serum 剞 1, _ and Japanese cerebral encephalitis virus free black spots 圄 Pan 丨 3 each rabbit serum was tested at a dilution of 1:1000 In the case of non-denatured, the strips of dengue 1, 2, 3, and 4 of the dengue separated by SDS PAGE were banded with JE virus antigen + control strips of cells not subjected to C6/36 5 by mBN07 vaccine Immunized rabbits were induced to recognize the true dengue virus 6 on it. Figure 8 shows that each rabbit immunized serum reacted with NS1 from dengue serotypes 1, 3 and 4 and reacted with Japanese encephalitis immune dots. 7 3. Conclusion ^54560 Antibody to serotype 2 NS1. - A high immune response was observed when the endpoints in the immunoblot analysis and ELISA were 1:10 -4 and 1:10 β. The antibody that was elicited in the rabbit reacted with all other dengue serotypes (1, 3 & 4). One such antibody also cross-reacts with NS1 from a heterologous virus such as JEV. Example 3: Growth kinetics of MVA-BN vector virus in selected cell lines, replication in vivo, and immunological data. As indicated in the specification section, the DNA of the present invention is preferably inserted into MVA- A carrier of BN cancer virus or a derivative thereof. The following examples describe MVA-BN in more detail. The disclosed embodiments allow those skilled in the art to recognize mva-BN and its derivatives. Growth kinetics of I cell lines: To characterize the MVA-BN, the growth kinetics of this variety were compared to other MVAs that have been characterized. This experiment was carried out by comparing the growth kinetics of the following viruses in the original cells and cell lines listed: MVA-BN (virus reserve #23, 18.02. 99 untreated, titrated into 2&gt ; 107X 107 TCIDso/ml); MVA characterized by Altenburger (US Patent No. 5, 185, 146) and additionally referred to as MVA-HLR;

MVA (passage 575) characterized by Anton Mayr (Mayr et al., [1975] Infection 3; 6-14) and otherwise known as MVA-575 (ECACC V00120707); and 37 1354560 in International Patent Application PCT/EP01 The characteristic MVA-Vero (viral reserve, passage 49, #20, 22.03.99 untreated, titrated to 4, 2 X 107 TCIDso/ml) is depicted in /02703 (WO 01/68820). 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 a 6-well-dish 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 〇 2 at near 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 is 5 X 104 TCIDso and this is called input. The cells were then washed three times with DMEM, and finally 1 ml of DMEM, 2% FCS was added and the plate was left at 37. (:, 96 hours (4 days) at 5% C〇2. The infection was terminated for titration analysis by freezing the plate at -80 ° C. 2. Titration analysis (specific with vaccine virus) Antibody immunostaining) To quantify the number of viruses, test cells (CEF) were seeded on 96-well-plate at a concentration of 1 X 1〇4 cells/well, RPMKGibco, Cat. No. 61870-010), 38 1354560 • · 7% FCS, 1% antibiotic/antimycotic (Gibc〇, Cat No. 15240-062), and incubated at 37 ° C, 5% COz to overnight ❶ will contain the infection of the 6-well - The plate was frozen/thawed 3 times and RPMI growth medium was used to release ΗΓ1 to 1 (T2. The virus dilution was dispersed onto the test cells and incubated at 37 ° C, 5% C 〇 2 for 5 days to allow CPE ( Cell morbidity) development. The test cells were fixed (acetone/methanol • 1:1) for 10 minutes' washed with PBS and placed in incubation buffer at ι:ι〇〇〇, with multiple vaccinia virus-specific antibodies. Cultivate together 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) at 1:1000 in incubation buffer was added. , lasted for 1 hour at room temperature. Wash again twice with PBS, and with the staining solution (10 ml PBS + 200 μΐ in 100% ethanol in 〇- • dimethoxybenzidine saturation solution + 15 μΐ freshly prepared Η2〇2) Incubated to observe brown spots (2 hours). 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 the Kaerber equation is used to calculate the titer (TCID50-based Φ 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 104 TCID5.), which is expected to be tolerant to MVA, and on the other hand, Replication 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 1354560 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 shows that all viruses are appropriately amplified in CEF (chicken embryonic fibroblast) cells as expected because this is a cell strain that is 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 10 fold input. Compared to the others, only MVA-BN was found to be not properly amplified in these cells, i.e., only a 2-fold increase over the input. : Regarding the replication in CV1 cells (kidney kidney cell strain), it was found that: MVA-BN was greatly reduced in this cell line. It shows a 200-fold reduction below 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, which has a 5-fold increase above the input. The most important thing is to compare the growth kinetics of various viruses in human cell lines. Regarding the efficient replication in 143B cells (human bone cancer cell lines), it was shown that MVA-Vero was the only one showing amplification higher than the input (3-fold increase) β all other viruses did not amplify higher than the input, However, there is a big difference between MVA-HLR and MVA-BN and MVA-575. The MVA-HLR system "wide range" (less than 1 fold reduction in input), such as MVA-BN shows minimal reduction (less than 40 300 times reduction of input), followed by MVA-575 (below input) 59 times 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 was well amplified in this cell (a 55-fold increase over the input). Both MVA-Vero and MVA-575 were shown to be amplified in this cell line (1.2 and 1.1 times higher than the input, respectively). However, MVA-BN is the only reduction in presentation (a 5-fold reduction from the input). The conclusions 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 included in Table 1) Medium), MVA-BN showed an extreme decrease in human cell lines. It further shows an amplification ratio of about 〇·0 in 143B cells; an amplification ratio of about 〇.4 in HeLa cells; and an amplification ratio of about 0.22 in HaCat cells. In addition, 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). The ratio of MVA-575 shows a similar quantitative curve to mva-BN, but not like MVA. -BN-like reduction" MVA-HLR amplifies well in all test cells except 143B cells. Therefore, it is considered to be sufficient for replication in all tested cell lines (except 143B cells). In some cases, its amplification in human cell lines (HeLa) is even better than that of a tolerant cell line (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. 3. 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 charge and is unable to produce mature 8 and 1' cells, so it is severely immune to abandonment and is very resistant to replication. 107 pfu of M VA-BN, MVA-HLR or MVA572 To immunize (ip) 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. At the time, there is a common phenomenon of serious viral infection in the main organs, and MVA (108 pfu) can be found again from the ovary by standard spot analysis. Conversely, the same dose of MVA-BNC corresponds to the registered variety. ECACC V00 083008) Rats after vaccination survived for more than 90 days, and no mva can be re-discovered from organs or tissues. 42 1354560 • · When data from both in vitro and in vivo were obtained together, the study showed that 'MVA-BN is more than the parent and Commercial MVA_HLR varieties are more severely reduced. 4. Different varieties of MVA are also different in stimulating immune response. Copying sufficient varieties of vaccinia induces immune response in mice, while high doses are fatal. Although MVA is highly reduced And there is a reduced 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. In order to determine whether the difference 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 After 4 days of stimulation, the titer determined in the ovarian vaccinia is reduced, and this allows quantitative evaluation of MVA of different doses and different varieties. Death Stimulation Model Immune (ip) 6-8-week-old female BALB/c (H-2d) mice with specific pathogens at different doses (102, 104 or 10 BTCID5〇/ml) of MVABN or MVA 575 (n=5) MVA-BN and MVA-575 have been propagated on CEF cells and have been purified by sucrose® and formulated into 7.4 in Tris. After three weeks, the mice received an increase in the same dose of MVA and MVA varieties for two weeks. Then, lethal stimulation (ip) is carried out with a variety of vaccinia. 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 - $ consolation. Protection can be measured by reducing the titer in the ovary as determined by standard spot analysis after 4 days of stimulation. Here, the mouse was sacrificed on the 4th day after the stimulation, and the ovaries were removed, homogenized in PBS (lml) and virus titer was determined using standard plaque analysis using VER0 fineness (Thomson et al., 1998, 43 1354560

Immunol. 160: 1717). After 4 days of stimulation, the mice immunized with either MVA-BN or MVA-575 at 104 or 10 BTCID5D/ml were completely protected by a 100% reduction in ovarian rVV titers (Fig. 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 lfTTCIDw/inl were unprotected' evaluated as high ovarian rVV titers (mean 3. 7 x 107 pfu +/- 2.11 x lO7). In contrast, mice vaccinated with the same dose of MVA-BN induced a significant decrease (96%) in terms of high ovarian rVV titers (mean 21 21 xi〇7 pfu +/- 〇. 287 xlO7). Rats in the control group receiving placebo had an average viral titer of 5. 11 xl07 pfu (+/- 3.59 xlO7) (Figure 2). Both MVA strains induced a protective immune response against lethal rw 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 initial-push-inoculation therapy 5.1.: Production of antibodies against MVA after inoculation of mice with different smallpox vaccines The efficacy of MVA-BN was compared 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 of smallpox. In addition, before the vaccines of both MVA_BN and MVA 572 were compared, the cuts were followed by Elstree. Each group used 44 1354560 with 8 BALB/c mice, and all MVA (lxl〇7 TCIDse) 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 induced by these different vaccines or therapies is measured in animals before stimulation. Neutralizing antibodies, T cell proliferation, cytokine products (IFN-γ and IL-4) and ΐρΝ_γ assays produced by T cells were used. The level of T cell response induced by MVA-βΝ is generally equivalent to its 2 MVA and bovine cancer virus when measured by ELIspot, showing bio-equivalent. After different vaccination therapies, the weekly titer of antibodies against MVA showed that the rate and size of the antibodies were significantly increased by MVA-BN vaccination compared to other inoculation methods (Figure )). · Indeed, compared to mice vaccinated with MVA 572, when vaccinated with MVA-BN, the antibody titer against MVA was evident at weeks 2, 4, and 5 (1 week after the 4th week) Higher (P>〇.05). After the booster vaccination in the fourth week, the antibody titer in the group was significantly higher than that of the single vaccination with the vaccinia strain Elstree or Wyeth. These results clearly show that compared to the typical single-vaccination with traditional bovine cancer strains (Elstree and Wyeth), two of the mva-catch vaccinators induced a more favorable antibody response, and from the region ^ 5 confirms that this finding is more immunogenic than other MVA strains. 5·2·. In the heart-stimulation model, mva's initial and push-up therapy produces the same level of protection as DNA-primary MVA-push therapy, which will be used to generate MVA primary-push-up therapy for active CTL response. Compared with the initial report of the DNA that has been reported to be the best. Different therapies were evaluated using a murine polyhedral structure encoded by a dirty vector or MVA BN and the CTL induced levels were compared at 45 1354560 ELISP0T, while the measured reaction activity was provided as a stimulus after influenza stimulation. The extent of protection. Results The DNA plastids encoding murine polyhedra (10 CTL epitopes, including influenza, ovalbumin) have been previously described (Th〇mson et al., 1998) Imnumol. 160: 1717) β This murine polyhedron was inserted The deletion position II of MVA_BN was grown in CEF cells, stored as sucrose and formulated into Qius 7.4 in Tris. Vaccination Steps In the current study, male BALB/c (H-2d) mice, 6-8 weeks old without specific virus, were used. Five mouse populations were used for ELISP0T analysis, and six 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 boosts. In the case of DNA inoculation, the mice were anesthetized and then anesthetized with 50 endotoxin-free plastid DNA (in 50 μL of PBS). The primary immunization used was performed by intravenously administering 1 〇7 pfu of MVA-BN per mouse or subcutaneously administering 1 p7 pfu4 10*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, 'in the use of influenza CTL epitope TYQRTRALV, P. Berghei epitope peptide (SYIPSAEKI), giant cell virus peptide epitope (YPHFMPTNL) and / or LCV wins Two weeks after the final boosting of the peptide epitope (RPQASGVYM), a standard ELISP0T analysis was performed on splenocytes (Schneider et al., 1998, Nat. Med. 4; 46 1354560 » 397-402). In the stimulation experiment, the mice were anesthetized, and the mice were infected with a secondary-lethal dose of the commonly used influenza virus, Mem71 (4.6 x x 5 5 fufu in 50 ml of PBS). On day 5 post-infection, the lungs were removed and the virus titer in duplicate was determined in Madin-Darby's dog tubular cell line using standard epidemic plaque assay. RESULTS: The DNA vaccine alone was used, and the CTL lineage induced by the 4H-2 d epitope encoded by the mouse polyhedron was insufficient, and the epitope of p. Berghei (SYIPSAEKI) ® and lymphocytic plexus meningitis virus (RPQASGVYM) were used. Both can only detect a small amount of reaction. Conversely, using DNA initial MVA push-up therapy (subcutaneous administration of 107 pfu MVA-BN), the induced CTL was significantly higher, for SLY (8_' fold increase) and for RPQ (3-fold increase), and also Observable for rodents

- The reaction of the third epitope of the giant cell virus (YPHFMPTNL) (Fig. 3A). However, subcutaneous administration of 1〇7 pfu mva_bn in the same initial ascending therapy induces the same response as DNA using MVA-BN (Fig. 3A). Surprisingly, when immunized with one MVA-BN (107 TCID50), there was no significant difference in the number of CTLs induced by the three epitopes, indicating that the second immunization with MVa_bn would not be apparent Increase the CTL response. It has previously been shown that subcutaneous administration of 1〇7 pfu MVA is the most inefficient route to virus concentration when inoculated with MVA from other strains, especially in comparison 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 the 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. %). 47 1354560 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. Moreover, unlike MVA-BN administered subcutaneously at 7 pfu, immunization with MVA-BN administered intravenously with pfu and subcutaneous administration of i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 ' Kalayanaroo 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 'flavor virus. The Virus, Fourth Edition, edited by David M Knipe and Peter M Howley. Published by Lippincott Williams and Wilkins, Philadelphia. Page 1043-1125

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. Initiation of protein synthesis by the 5' untranslated region of the ribosome into the cerebral myocarditis virus RNA J. Virol., 63:1651-60 48 13545 (50)

Lindenbach BD and Rice CM., 2001, flavivirus and its replication. In the field of viruses, 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 yellow fever encephalitis decision: site. 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 Br i an WJ Mahy and H i 11 ar 0 Kangro. Academic Press. 1996 Molecular Virology: A Practical Introduction. Edited by AJ Davison and RM Elliott. 49 1354560 Practical Starter Series. IRL Press at Oxford University Press· Oxford 1993. Chapter 9: Gene Expression of Vaccinia Virus-Loaded Genes Current rules of molecular biology. Issuer: John Wiley and Son Inc. 1998. Chapter 16, Section IV: The performance of proteins using vaccinia virus vectors in mammals. Anti-Think, Laboratory Manual. Edited by Har 1 ow and Dav i d Lane. Published by Cold Spring Harbor Laboratory, 1988. I: BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a diagram showing the dengue heat NGC variety "signal sequence + NS1" 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 nuclear wicking acid and the amino acid in Fig. 1A correspond to 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 p-acid sequence of the NS1 cassette in plastid pAF7 is continued to 'PCR-amplify the promoter linkage of this cassette with OBN345 and 〇BN338. The nucleotides in Figure 1C The amino acid sequence corresponds to SEQ ID NO: 7. This 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 data 庳 accession number AF038403 amino acid 776 to 1127). Value above zero = hydrophobicity 50. Below: Kyte-Doolittle hydrophilic dot map 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 (amino acid) 748 to 775). 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. Figure 2: "pox virus promoter + signal sequence + milk thistle" performance cardinal acid sequence. 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: 1. 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 Xho I position of the phΒΝΧ〇7 terminus (the blunt end of the flat mouth) to produce 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: Hin of MVA (Gene Library U94848) (i III map showing six missing addresses of MVA (-J-=missing junction).” ppr+NPT II + IRES+EGFP+PPr+ NSl" cassette is inserted into the deletion position of MVA. PPr = poxvirus promoter, NPT II = neomycin resistance gene (protein coding sequence), IRES = ribosome binding site and NS1 = signal sequence + dengue 2 NGC The NS1 protein coding sequence of the variety. 1354560 Fig. 4: Immunoblot map showing mBN07 and true dengue virus serotype 2 with uninfected cell control group, which was probed against monoclonal antibodies against dengue virus NS1 protein. The arrow refers to the natural NS1 dimer, while the arrow to the right refers to the NS1 monomer, which is viewed after the sample is boiled. 2ME = 2 mercaptoethanol. Figure 5: Serum diluted to 1:200 was tested, no reduction and no In the case of heating, the immune dot spots of the dengue virus serotype 2 antigen isolated by SDS PAGE were on the control strip of uninfected C6/36 cells. Band 1 = dengue virus serotype 2 antigen strip Band; strip 2 = control strip; a = Pre-epidemic serum, and b = serum after vaccination. Figure 6: Serum after immunization is titrated to 1:1000, 1:2000, 1:4000, 1:1 (Γ4, 1:1 (Γ5, 1 :1 (Γ6, and was tested on the control strips of dengue 2 virus antigen and the control strip of uninfected C6/36 cells after SDS PAGE separation without reduction and without heating. : For rabbits #1 and #2, the NS1 band diluted 10–4 could not be observed on the scanned photograph, but 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. : Each rabbit serum diluted to 1:1000 was tested on a strip of immunological dots of dengue 1, 2, 3, 4+ JE virus antigens separated by SDS PAGE without reduction and without heating. 52 1354560 control strips of uninfected C6/36 cells. Note: For NS1 bands with dengue 3 and 4 with rabbit #2 serum, although not The photographs are observed, but the actual bands 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 288135*4560 • « ggc att tgt gga ate ege tea gta aca aga ctg gaa aat ctg atg tgg Gly 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 Lys Gin lie 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 Lys Leu Thr lie Met Thr Gly Asp lie Lys Gly lie Met Gin Ala Gly 115 120 125 336 384 aaa ega tet ctg cag ccc cag ccc act gag ctg aag tat tea tgg aaa Lys Arg Ser Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys 130 135 140 432

Aca tgg ggc aaa geg aaa atg etc tet aca gag tet cat aac cag acc Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His Asn Gin Thr 145 150 155 160 480 ttt etc att gat ggc ccc gaa aca gca gaa tgc ccc aac Aca aac aga Phe Leu lie Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg 165 170 175 get tgg aat teg ctg gaa gtt gaa gac tat ggc ttt gga gta ttc acc Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr 180 185 190 528 576 acc aat ata tgg eta aag ttg aga gaa aag cag gat gta ttc tgc gac Thr Asn lie Trp Leu Lys Leu Arg Glu Lys Gin Asp Val Phe Cys Asp 195 200 205 624

Tea aaa etc atg tea geg gee ata aaa gac aac aga gee gtc cat gee Ser Lys Leu Met Ser Ala Ala lie Lys Asp Asn Arg Ala Val His Ala 210 215 220 672 gat atg ggt tat tgg ata gaa agt gca etc aat gac aca tgg Aag ata Asp Met Gly Tyr Trp lie 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 Glu Lys Ala Ser Phe lie Glu Val Lys Ser Cys His Trp Pro Lys Ser 245 250 255 720 768 cac acc etc tgg agt aat gga gtg tta gaa agt gag atg ata cc His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met lie lie Pro 260 265 270 3 816 1354560 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 275 280 285 cat aca caa aca gca gga cca tgg cat eta ggt aag ett gag atg gac 912

His Thr Gin Thr Ala Gly Pro Tip 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 gee tet gga aaa etc ata 1008

Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu lie 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 tee ttg gtc aca gee 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 View 2 <400> 6

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 He Gin Lys Ala His Glu Glu 4 1354560 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 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 He Lys Asp Asn Arg Ala Val His Ala 210 215 220

Asp Met Gly Tyr Trp He 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 He lie 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 5 1354560 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> 7 <211> 1296 <212> DNA ' <213> Dengue Noodles 2 <220><221> CDS <222> (50)..(1189) <220><221>Introduction_Combination卩<222> (12)..(32) <220><;221>Introduction-integration<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 A Sn 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 6

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 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 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 gac ate aaa gga ate atg cag gca gga aaa ega tet lie Met Thr Gly Asp lie Lys Gly lie Met Gin Ala Gly Lys Arg Ser 120 125 130 ctg cag ccc cag ccc act gag ctg aag tat tea tgg aaa aca tgg ggc

Leu Gin Pro Gin Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly 135 140 145 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 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 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 215 220 225 tat tgg ata gaa agt gca etc aat gac aca tgg aag ata gag aaa gee

Tyx Trp lie Glu Ser Ala Leu Asn Asp Thr Trp Lys lie Glu Lys Ala 230 235 240 tet ttc ate gaa gtt aaa age tgc. cac tgg cca aag tea cac acc etc 7 1354560

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 ccc 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 view 2 <400>

Met Asn Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly 1 5 10 15 8 in 1354560 •

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 100 105 110

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 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 He lie Pro .260 265 270 9 1354560

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

<210> 9 <211> 1227 <212> DNA <213> Dengue virus type 2 <220>

<221>Starter <222> (5)..(48) <223> Minimal disease starter 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 Asn 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 gcc ata aaa

Lys Gin Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala lie Lys 11 7831354560 205 210 215 gac aac aga gcc gtc cat gcc gat atg ggt tat tag ata gaa agt gca 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 gcc tet ttc ate gaa gtt aaa 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 Et t t t ag ag g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g 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 His Asn Tyr Arg Pro Gly Tyr His Thr Gin Thr Ala Gly Pro Trp His 285 290 295 eta ggt aag Tett gag atg gac ttt gat ttc tgc gaa gga acc aca gtg Leu Gly Lys Leu Glu 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 Va l Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr 315 320 325 act gcc tet gga aaa etc ata aca gaa tgg tgc tgc ega tet tgc aca 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 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 Glu lie Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu 365 370 375 gtc aca gcc tag Val Thr Ala 380 831 879 927

975 1023 1071 1119 1167 1215

<210> 10 12 1227 1354560 '^ <211> 380

<212> PRT ' <213> Dengue virus type 2 <400> 10

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 lie 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 1354560 , 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

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)

1354560 * 妙.r V Drop (more) Original Patent Application No. 99138145 A. Please apply for the patent scope: 1. A pharmaceutical composition for treating or preventing flavivirus infection. The composition comprises a poxvirus vector carrying a DNA comprising a cassette which comprises a transcriptional regulatory element and a nucleic acid sequence encoding a complete flavivirus NS1 protein or An antigenic epitope thereof, and wherein the flavivirus infection is an additional/additional flavivirus and/or flavivirus serotype that is not derived from the nucleic acid sequence or the N S1 protein or antigenic epitope thereof Infection, and 10 presuppose that the poxvirus vector is not as follows: an epidemic viral vector containing a DNA containing the expression card IE, the expression concealment comprising a transcriptional regulatory element and a sequence encoding a complete The flavivirus NS1 protein or one of its antigenic epitopes and selectively encodes another peptide/protein that is not a complete flavivirus 15 E-egg The virus is a modified bovine virus, Ankera strain (MVA), which has the following characteristics: (i) can replicate in chicken embryonic fibroblasts (CEF), but in human keratinocyte cell line HaCat, Human osteosarcoma cell line 143B and human cervical adenoma cell line HeLa have no reproductive replication energy 20 (') cannot replicate in a mouse model that does not produce mature B and T cells; and the premise is that the pharmaceutical composition is not as follows Said: ~ a pharmaceutical composition for the treatment or prevention of flavivirus infection, comprising a modified bovine cancer virus An 1 No. 38〗 No. 45 Patent Application Patent Revision Amendment This revision period: September 27, 100 a VAKura strain (MVA) virus vector, the river VIII virus vector carrying a performance cassette, wherein the expression cassette contains a transcriptional regulatory element and a nucleic acid sequence, and the nucleic acid sequence is browned into a dengue virus serotype 2 The intact flavivirus NS1 protein or an antigenic epitope thereof and selectively encodes another peptide/protein which is not a complete kappa 毋E-protein, and wherein Flavivirus infection blood out type 1, Dengue virus serotype 3 gray, infection by the dengue virus selected from dengue serotype 4 virus, said encephalitis virus, West Nile virus, and yellow caused by the virus. 2. If you apply for a patent scope! A pharmaceutically acceptable composition for use in the treatment or prophylaxis - an animal to additionally combat against a flavivirus and/or flavivirus serotype from which the NS1 protein or antigenic epitope thereof is derived. 3. The pharmaceutical composition according to claim 1 or 2, wherein the flavivirus is a mosquito-borne flavivirus. 4. The pharmaceutical composition of claim 3, wherein the mosquito-transmitted flavivirus is a dengue virus. 5. If the pharmacy composition of the fourth item of the full-time division is applied, the dengue virus system is dengue virus serotype 2. 6. If the pharmacy group of the NS1 protein or its antigenic epitope, which encodes the flavivirus, is preceded by an ATG codon and a coding glycation sequence. The sequence, and wherein the coding sequence is terminated by a translation termination Mimma. 7. For the pharmaceutical composition of claim 1 or 2 of the patent (4), wherein the transcriptional regulation is 1354560, and the patent application scope of the patent application is amended. The date of revision is: September 27, 100, the factor is a poxvirus promoter. . 8. The pharmaceutical composition according to claim 1 or 2, wherein the poxvirus vector is freeze-dried. 9. The pharmaceutical composition according to claim 1 or 2, wherein the poxvirus 5 is a modified vaccinia virus Ankera strain (MVA). 10. The pharmaceutical composition of claim 1 or 2, wherein the NS1 protein or antigenic epitope thereof is derived from a dengue virus serotype, and wherein the composition protects a body to at least At least two infections caused by dengue virus serotypes. The pharmaceutical composition according to claim 1 or 2, wherein the composition protects one body against infection by all dengue virus serotypes. 12. The pharmaceutical composition according to claim 1 or 2, wherein the flavivirus serotype and/or flavivirus line from which the NS1 protein is derived is dengue virus serotype 2. 15. The pharmaceutical composition of claim 1 or 2, wherein the additional/additional flavivirus is selected from the group consisting of West Nile virus, yellow fever virus and sputum encephalitis virus. 14. The pharmaceutical composition according to claim 1 or 2, wherein the composition is supplied for initial vaccination or additional vaccination, or for initial vaccination and addition of 20 species. 15. The pharmaceutical composition according to claim 1 or 2, wherein the composition is formulated as a set, the set comprising the poxvirus vector, which is used for the first vaccination (primary vaccination) The second vial/container in the first vial/container is located in a second vial/container for the second inoculation (additional inoculation). Patent Application No. 99138145 Patent Application Revision This revision period: September of the following year In the middle of the 27th. 16. The use of a nucleic acid poxvirus vector for the preparation of a composition comprising a expression cassette comprising a transcriptional regulatory element and a nucleic acid sequence encoding a complete A flavivirus NS1 protein or an antigenic epitope thereof, which is used to treat or prevent an animal against additional/extra flaviviruses that are not derived from the nucleic acid sequence or the NS1 protein or antigenic epitope thereof And/or a flavivirus serotype, and provided that the poxvirus vector is not as follows: a poxvirus vector comprising a DNA comprising a cassette, the expression cassette comprising a transcriptional regulatory element and a sequence, the sequence Encoding a complete flavivirus NS 1 protein or an antigenic epitope thereof and selectively encoding another peptide/protein which is not a complete flavivirus E-protein and the poxvirus is The modified vaccinia virus Ankera strain (MVA) has the following characteristics: (i) can replicate in chicken embryonic fibroblasts (CEF) but is fine in human keratinocytes HaCat, human osteosarcoma cell line 143B and human cervical adenoma cell line HeLa have no reproductive replication capacity, (ii) cannot replicate in a mouse model that does not produce mature 6 and butyl cells; and the premise is that the composition is not as follows The pharmaceutical composition for treating or preventing a flavivirus infection, comprising a cowmark virus Ankera strain (MVA) virus vector, the MVA virus vector carrying a performance cassette, wherein the table 1354560 • · 99138145 Patent Application for Patent Application Amendment Revision Date: September 27, 100 10 20 The present cassette contains a transcriptional regulatory element and a nucleic acid sequence encoding a complete flavivirus NS1 protein of dengue virus serotype 2 or an antigenic epitope thereof and optionally encoding another peptide/ Protein, the other peptide/protein is not a complete flavivirus E-protein, and 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 infection caused by the West Nile virus's flavivirus. 17. The use of claim 16, wherein the flavivirus infection is additionally an infection of a flavivirus and/or flavivirus serotype derived from the nucleic acid or the N S1 protein or antigenic epitope thereof . 18. The use of claim 16 or 17, wherein the flavivirus is a mosquito-borne flavivirus. 19. For the use of claim 18, wherein the mosquito-borne flavivirus is a dengue virus. 20. The use of claim 19, wherein the dengue virus is a dengue virus serotype 2. 21. The use of claim 16 or 17, 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 glycated signal sequence, and wherein The coding sequence is terminated by a translation stop codon. 22. The use of claim 16 or 17, wherein the transcriptional regulatory element is a poxvirus promoter. 23. The use of claim 16 or 17, wherein the poxvirus vector is freeze-dried. 5 Patent Application No. 99138145, the scope of the patent application, the date of this amendment, · September, 2007, 曰24. 24. For the use of the scope of claim 16 or 17, wherein the poxvirus is a modified vaccinia virus Ankera strain (MVA). 25. The use of claim 16 or π, wherein the nsi protein or antigenic epitope thereof is derived from a dengue virus serotype and wherein the composition, agent or vaccine system protects a body to at least counteract Infected by at least two dengue virus serotypes. 26. The use of claim 16 or 17, wherein the composition protects the individual against infection by all dengue virus ash clear types. 27. The use of claim 16 or 17, wherein the flavivirus serotype and/or flavivirus derived from the protein is dengue virus serotype 2. 28. A flavivirus other than the scope of the patent application is selected from the group consisting of the additional/frontal encephalitis virus. , Luo Xiao virus, yellow fever virus and Japan π. If the patent application scope is 'cut for initial vaccination or recovery' - the composition is the supplier. 'Or the first vaccination and additional vaccination, such as the application for patent scope 16 or the production-set, where the composition is supplied for the 帛-cut I 3 H riding, the line is in one - The first vial/container for the second inoculation) is supplied. Second vial/capacity for human inoculation (additional inoculation) 31. For example, the scope of the patent application. Where the "use of the item" of the composition is a 1354560 Patent Application No. 99138145, the scope of application of the patent is amended. Date of revision: September 27, 100. 32. 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