JP2000507824A - Therapeutic recombinant poxvirus encoding myelin protein - Google Patents

Abstract

  (57) [Summary] Recombinant poxviruses encoding myelin proteins have been disclosed. The use of a recombinant poxvirus can elicit an immune response against myelin proteins. The method comprises introducing a sufficient amount of poxvirus to present the myelin protein to the immune system, expressing the myelin protein, and presenting the myelin protein to the immune system.

Description

DETAILED DESCRIPTION OF THE INVENTIONTherapeutic recombinant poxvirus encoding myelin protein   As part of the research expenses of the present invention, NIH subsidy No. NS30727 was contributed, Accordingly, the United States government has certain rights in the invention.Background of the Invention   Central nervous system (CNS) such as multiple sclerosis (MS) and Guillain-Barre syndrome Inflammatory desheathing is a disease that involves an immune response to the CNS autoantigen It is considered. Is it a disease model like experimental allergic encephalomyelitis (EAE)? By analogy, myelin proteins, especially myelin basic protein (MBP) That the immune response to thyroid plays a role in the development and progression of inflammatory desheathing disease Indirect evidence suggests. For example, in the case of human MS patients, typically MBP Responsive T cells are recovered from cerebrospinal fluid of MS patients (1, 2 and 3). MS patients Concentration of MBP-reactive T cells in cerebrospinal fluid (CSF) affected other nervous system disorders It is higher than the control concentration (2). MBP-reactive T cells are also present in MS foci Directly detected (3). In the case of EAE, active immunization with MBP or an antigenic fragment of MBP Or because disease can be induced by adoptive immunization of MBP-reactive T cells, BP is the major antigen of this disease (4,5).   MBP is a component of the myelin protein. MBP gene is a selective RNA Encodes several MBP related proteins by pricing (Lemki , G .; (1988), Mikoshiba, K .; (1991), Kamhol z, J.M. (1987)). Proteolipid protein (PLP), myelin-related Glycoprotein (MAG), myelin / oligodendrocyte glycoprotein (MO The gene sequences of other myelin proteins such as G) are also known (Linging). ton et al., Eur. J. Immunol. 23: 1364 (1993)).   EAE susceptible animals are a research model for human inflammatory desheathing diseases such as MS It recognized. EAE is typically whole myelin, MBP, the antigenicity of MBP Immunization with fragments or adoptive immunization with MBP-reactive T cells Thus, it can be triggered (12, 13, 4, 5). Rodents suffer from EAE Also recovers normally. This is because the immune system is cytotoxic T cells or anti-inflammatory It shows that disease is suppressed by supplying cytokines (6, 11). However, human MS is recurrent, distinct from most forms of rodent acute EAE And chronic diseases. Little is known about the factors responsible for human disease recurrence Not. Regulatory T cells in human diseases (eg suppressor T cells) The role of has not been well studied.   Primates Callithrix jacchus (C. jacchus, common Marmosets), either active immunization in whole white matter or MBP-reactive T cells Alternatively, EAE can be induced by adoptive immunization with clones (12-1). 4). While marmosets are a particularly advantageous model for studying inflammatory desheathing, One reason is that siblings with common parents adopt lymphocytes between genetically distinct individuals. This is because they share bone marrow chimerism that allows transfer. Mars with EAE In moset, the response of T cells and antibodies to MBP during disease onset Sex is detected, which can be observed in peripheral blood mononuclear cell (PBMC) specimens (13) .   Poxviridae virus (poxvirus) is for clinical and research purposes In many integration operations performed in And a vector for delivering gene products. Oh for several reasons A poxvirus of the genus Orthopoxvirus, in particular Vaccinore virus is used. The reasons are, for example, (a) eradication of smallpox (B) including cells specialized in antigen presentation. Infects a wide range of cells and produces inserted gene products (for example, foreign gene products). The gene product is a class I and / or class II M Being able to be processed based on its relationship to HC molecules, (c) some tumors Being used as a recombinant vaccine in the treatment of ulcers (Kantor, J (1992)).   Avipoxvirus like fowlpox, canarypox, swinepox Many other poxviruses, such as those belonging to the genus Suipoxvirus, Used as a vector for chitin and as a vector for intracellular expression of foreign genes. Have been.   Myelinators such as neural sheath proteins, especially MBP and MBP-related proteins There is a need for the development of vaccines that can induce immune tolerance to proteins.Summary of the Invention   We have determined that the intracellular nature of the myelin protein or its T cell-inducing epitope By using recombinant poxvirus for expression, vertebrate host It has been found that symptoms of inflammatory desheathing disease can be reduced or postponed. More specific Typically, recombinant poxvirus and myelin protein are co-expressed in cells. This allows the subsequent host immune system to substantially respond to myelin proteins. It was found that it became tolerant and helped reduce the symptoms of the disease or delay the onset. Fine The term intracellular expression refers to the process by which protein transcription and translation take place inside a host cell. Means to occur. The protein itself is maintained inside the cytoplasm, or Or transported to the nucleus, cell membrane, or other intracellular area or extracellular space. Can be   Recombinant poxviruses contain the myelin protein or its T cell-induced epidermis. At least one insertion site containing a DNA segment encoding a tope This DNA segment is operable with a promoter that can be expressed in the host. Noh is linked. The term “myelin protein” refers to MBP, PLP, M Concentric surroundings around axons such as OP and MAG Means the protein that constitutes the target oligodendrocyte. Preferred myelinta Proteins are MBP, MBP-related proteins and their T cell-inducing epitopes. You. Other proteins include MOP and MDP (myelodendritic protein). Quality).   Generally, the preferred method of the present invention involves the use of a sufficient amount to stimulate an immune response. Introduction of the first recombinant poxvirus vector into the host Presenting the protein to the immune system. In some cases, It is desirable to contact the host with at least one booster antigen as a "boost." Preferably, the first recombinant poxvirus is a pox of a different genus than "boost". Derived from a virus. For example, the first dose is recombinant from avipox Poxvirus, followed by another recombinant pox from Suipox. Boost with virus. The first recombinant poxvirus vector was Myeri DNA segment encoding a protein or a T cell-inducing portion thereof I have. As described above, the additional antigen may be immunologically associated with the first poxvirus, for example. Different poxvirus, typically different from the first poxvirus Added using a second recombinant poxvirus derived from the genus poxvirus You.   Alternative to expression of myelin protein by first recombinant poxvirus or In addition, additional myelin protein or a T cell-inducing fragment thereof is added. May be added. For this purpose, the adjuvant or the liposome Contacting a phosphoprotein with a host or encoding a myelin protein DNA directly or in the form of a virus inherent in a selective viral vector Contact host.   It is also an object of the present invention to provide a recombinant poxvirus DNA or recombinant poxvirus. Contains the encoded myelin protein A cell, preferably intracellularly expressed in an amount sufficient to elicit an immune response Is to provide a homogeneous cell population. The cell is preferably a virus or a tan. A cell that can support expression of a protein for at least 48 hours, for example, a mammal. , Bird, reptile or insect cell lines.   According to another feature, the object of the invention also relates to one or more of the objects disclosed in the invention. Of recombinant poxviruses, Therapeutic composition wherein the virus (s) are provided in a pharmaceutically acceptable carrier It is to provide. Therapeutic compositions may be used, for example, for inflammatory desheathing diseases such as MS. It can be administered to mammals, preferably humans, as a means of reducing or delaying the onset. Therapeutic compositions of the invention may be administered alone or, for example, Interferon, corticotropin (ACTH) and corticos for the treatment of MS Adjuncts to known therapeutic methods for treating inflammatory desheathing diseases such as teroids May be administered as Thus, dogs, cats, horses, rabbits, mice, pigs, Mammals and breeding habits such as cows, reptiles, gerbils, birds, sheep and goats Inflammatory shedding disease in captive animals such as prey or primates (eg, chimpanzees) Is also included within the scope of the present invention.   The term “inflammatory desheathing disease” destroys, damages or removes the myelin of nerve fibers Means some immunological response. In mammals, especially humans, such damages The wound confirms the diagnosis of inflammatory desheathing disease.   It is also an object of the present invention to generate an immune response against a myelin protein in a host. Is to provide a way to The method is   (A) containing a sufficient amount of myelin protein or its T cell-inducing epitope; Mainly contacting,   (B) at least one regular interval thereafter, with additional myelin protein Contacting the host with the protein or its T cell-inducing epitope.   Another object of the present invention is to provide a method for producing a myelin protein capable of eliciting an immune response. To provide. The method is   (A) myelin protein, preferably MBP, MBP-related protein or The DNA segment encoding the T cell-inducing epitope is operably linked. Avipox, preferably fowlpox, canarypox, capripo Synthesizes recombinant poxviruses such as pox or swipox like swinepox To do,   (B) cells that support gene expression by the virus, preferably skin cells or Is a recombinant poxvirus that can infect muscle cells under mammalian host conditions. Preferably a step of introduction into a human,   (C) Myelin protein is injected intracellularly so that an immune response is elicited in the host body. Expressing and presenting properly.   The method also includes detecting the onset of an inflammatory desheathing disease in the host To produce cells (or their lysates) that can be used to determine the progress of Useful for The cell properly presents the myelin protein and thus Recognized by isolated T cells. Killing by T cells can be Detection by standard methods such as measurement of chromium released from cell labeled cells it can.   In another embodiment of the present invention, the method further comprises sensitizing the recombinant poxvirus. Isolating the stained cells; and expressing the myelin protein intracellularly in the infected cells. As an immunogen to produce antibodies that bind to the substance, preferably monoclonal antibodies, Using cells in the form of whole cells or cell fractions. Of course, antibodies It may bind to a T cell-inducing epitope on the Erin protein. Such antibodies Can be readily produced by those skilled in the art by conventional immunological techniques.   According to yet another embodiment of the present invention, the method further comprises isolating cells from the infected host. (Or the step of isolating the infected cells themselves) and the inflammation of the mammal, eg, a human Replace these cells with whole cells or cells to reduce or delay the onset of Spore fraction Eliciting an immune response in the second host by use in form .BRIEF DESCRIPTION OF THE FIGURES   1A and 1B show vaccines of vAbT249 (control) and vT15 (MBP). Inoculated with C. Jacchus marmoset showing two clinical courses of EAE It is a graph.   A, B, C and D in FIG. 2 show vT15 (MBP) (A and C in FIG. 2) and vA WM immunized C. vaccinated with bT249 (control) (FIGS. 2B and D). ja 4 is four graphs showing the proliferative response in PBMC of cchus marmosets. Figure 2A and 2B show in vitro stimulation with MBP (50 μg / ml). 2C and 2D show stimulation by NYCBH vaccinia antigen (2 μg / ml). .Detailed description of the invention   Poxviridae viruses are known cytoplasmic viruses. Therefore, Genetic material expressed by light recombinant poxviruses is typically in the cytoplasm. It is maintained and cannot be accidentally incorporated into host cell genes. In addition, pox Because of the large genome of ils, it has a broad spectrum containing many foreign genes. To deliver the genetic material (ie, to act as a multivalent vector) ) Can be used easily.   The poxviruses disclosed herein are typically those of the Poxviridae family. Preferably avipox, capripox, orthopox, entmopox and And Suipox. Preferred Avi Poxviruses include fowlpox, canarypox, pigeonpox, turkeypox, and cramppox. Especially good A better avipox is fowlpox. Preferred orthopoxviruses are waxy Near, cowpox, mouse pox (Ectromelia), rabbit pox, raccoon pox, and Including monkey pox. A particularly preferred orthopox is vaccinia. Preferably, Suipox is a swinepox and Capripox is a sheep or goat pox Ruth.   When the host is a human, pox has a restricted host range so that the host is not a human. It may be preferable to use ils. Avipo Box, capripox, entmopox or suipox for human use. Can easily respond. Poxviruses containing humans in the host range In this case, poxvirus should be used until it loses toxicity. Sufficient dilution (ie, attenuation) can address such limitations. An example See, for example, the NYVAC strain of vaccinia described in US Pat. No. 5,494,807. It is good to shine. The contents of the patent are incorporated herein by reference. See also the MVA strain described in US Pat. No. 5,185,145. The The contents of patents are also included in the present invention by reference. The immune system is damaged Use of vectors consisting of such attenuated poxviruses Particularly preferred.   Although not as preferred as poxviruses, other recombinant viruses (and the Virus-derived DNA) are also included in the scope of the present invention. These viruses Herpes virus, polyoma virus (eg, SV40 and polyoma ), Adenovirus, adeno-associated virus, etc. RNA viruses such as retroviruses and picornaviruses, for example polio Virus, Sindbis virus, Venezuelan equine encephalitis virus. Other c Ils includes frog virus, African swine fever virus There is Irus.   Choose a parent virus that is not toxic to the putative host animal Must. For example, a more highly attenuated virus, a virus that does not replicate, etc. Can be used for the host.   In a preferred embodiment of the invention, the recombinant poxvirus is infectious with the host. It shows low replication efficiency in the vesicle (ie, target cell). Generally, the replication efficiency is, for example, Cells that infect the virus and then tolerate the amount of progeny virus produced by the infected cells. Conventional methods such as quantification by titration above (eg, viral plaque assay) It can be measured by an illogical cell culture assay. After being produced in this way The amount of progeny virus is expressed as an amount relative to the total number of cells used in the experiment. According to the invention The preferred poxvirus used is preferably about 1 / cell / cell on average. Produce no more than 0 productive (ie, infectious) progeny, and more preferably, no more than about 1 productive progeny Progeny, more preferably less than 0.1 progeny per cell. Productive progeny The term refers to progeny that infect and replicate in permissive host cells.   Poor poxvirus replication efficiency and low integration into the cytoplasm By virtue of being sex, the recombinant pocks produced from these poxviruses Viruses do not sustain replication and infection of other cells. Thus vectors and traits Converted cell Does not adversely affect cells of the host animal remote from the target cell. Follow To produce attenuated strains of virulent poxvirus If the viral toxicity has been attenuated by genetic mutagenesis, More virulent poxvirus to prepare light recombinant poxvirus Can be used. See the aforementioned U.S. Patent No. 5,494,807.   Although we do not deny the possibility of interpretation by other theories, we have It is believed that the lus vector elicits a CD8 + response. Myelin coded The antigen may be myelin antigen-specific CD4 cells, possibly via a CD8 + cell-mediated response. Specifically triggers down regulation of Alternatively, an inhibitory site by regulatory T cells Either secretion of cain or both may occur.Preparation of recombinant poxvirus and vector   A recombinant poxvirus containing a heterologous DNA sequence encoding a foreign protein The basic techniques for making are well known in the art. For example, recombinant pox One method for making ills is to flank the DNA sequence in the donor plasmid. Contact Utilizes homologous recombination between an ils DNA sequence and a homologous sequence present in the parent virus (Macckett et al., Proc. Natl. Acad. Sci. USA 79: 7415-7419 (1982)). No. 5,093,258. See also the method for producing recombinant fowlpox virus. The contents of this patent are for reference only. Therefore, they are included in the present invention.   Another technique for producing recombinant poxviruses involves inserting heterologous DNA. Naturally or artificially introduced into the parent viral vector to A restriction endonuclease cleavage site is used. Also included in the present invention by reference No. 5,445,953.   Restriction digestion, ligation, transfection, transformation, agarose and poly Acrylamide gel electrophoresis, DNA sequencing, electroporation Such recombinant DNA manipulation methods have heretofore been known in the art and have been described in the literature. (Eg, Sambrook et al., In Molecular Clonin). g: A Laboratory Approach (1988); Ausub el et al., Current Protocols in Molecular B iology, Green Publishing Associates and Wiley Inte rsciences (1993)).   Typically, a heterologous DNA sequence to be inserted into a poxvirus is Sumid, bacteriophage or virus, such as pBR322 or pUC 19 may be located on a cerebral fungal plasmid. When a donor plasmid is selected In some embodiments, the plasmid preferably has one or more origins of replication (ORI). Detectable markers, such as antibiotics that grow in E. coli (eg, ampicillin). Or tetracycline) resistance gene. I'll explain more fully below As such, it typically associates with a poxvirus DNA section adjacent to the insertion site. The same DNA sequence is adjacent to the heterologous DNA.   Alternatively or additionally, a heterologous DNA gene sequence to be inserted can be replaced by a suitable promoter. May be combined with the Next, the promoter gene linkage is activated in the plasmid. Arrange as possible. That is, it is adjacent to the pox DNA region that is the desired insertion region. Place the DNA homologous to the DNA sequence adjacent to both ends of the promoter gene sequence. Place. When using the parent poxvirus vector, the pox promoter is usually Use The obtained recombinant plastic The Escherichia coli is then used to transform the Escherichia coli to select for the recombinant plasmid to replicate. Grow cells under alternative conditions. Next, the replicated plasmids are To purify. Of course, separate plasmids and eukaryotic cells for growing heterologous DNA Sex vectors (eg SV40 growing in green monkey cells) may be used ( See Sambrook et al., Supra).   A preferred method for producing the recombinant poxvirus of the present invention is to use a heterologous D Inserting the NA into the poxvirus by genetic recombination. For example The recombinant plasmid containing the heterologous DNA sequence to be inserted is replaced with chicken embryo fibroblasts. In such a cell culture, a poxvirus such as fowlpox virus or swinepox virus And transfect with it. Homologous pox DNA in plasmid and virus As a result of recombination with the genome, the presence of a promoter gene construct in the genome A modified recombinant poxvirus is obtained. Preferably, the recombination site is It is a site that does not affect the viability of ills.   As described above, the obtained recombinant virus substantially affects the virus viability. Insert the gene into a region (insertion region or site) in the virus that cannot be obtained. “Wil The term "viability" is a group Infectious virus particles within about 48 hours in the permissive host cell when the modified poxvirus Means the ability to produce Of course, in some situations the insertion will Effect, for example, the recombinant pox May attenuate ills. See the description below for the reason. No.   If it is not desired to substantially affect virus viability, one of ordinary skill in the art By testing segments of the viral DNA of Regions that support genetic recombination without significant impact on DNA . One readily available region that exists in many viruses is thymidine kinase. (TK) gene. This gene contains virtually all pox viruses tested. (Repolipoxvirus; Upton et al. (1986) (Shoop fibroma virus): Capripox virus; Gershon et al. (1 989) (Kenya sheep-1): orthopoxvirus; Weir (1983) (vaccinia); Esposito et al. (1984) (monkeypox Ruth and smallpox Hruby et al. (1983) (vaccinia); Kilpatrick. (1985) (Yaba monkey tumor virus): Avipox virus; Bin ns et al. (1988) (fowlpox); Boyle et al. (1987) (fowlpox); Schni tzlein et al. (1988) (fowlpox, quailpox): Entmopox (Lytvyn) (1992)).   In vaccinia, in addition to the TK region, virus viability is not substantially affected. Other insertion regions include, for example, a HindIII M fragment.   In fowlpox, in addition to the TK region, other insertion regions are, for example, BamHIJ flags. (Jenkins et al. (1991), EPO Application No. 0308220A1) EcoRI-HindIII fragment, EcoRV-Hind III fragment, BamHI fragment and HindIII fragment (Calvert et al. (1993); Taylor (1998); Spehner (1990) and Boursnell et al. (1990)).   In swinepox, the preferred insertion site is the thymidine kinase gene region including.   For successful expression of the insert DNA by the recombinant poxvirus, the insert Operable on heterologous DNA, in addition to the requirement that heterologous DNA be inserted into the region The presence of a promoter linked to is required. That is, the heterologous DNA and the promoter And the poxvirus exist in an appropriate spatial relationship that supports the transcription of the heterologous DNA. Must be present. The promoter is located upstream from the DNA to be expressed Must be arranged to be. Promoters are known in the art and The choice can be made easily based on the desired host and cell type. For example, Pox Will Used a promoter derived from the poxvirus promoter. You. These are like Vaccinia 7.5K or 40K or FPV C1. Based on natural promoters such as the fowlpox promoter Artificial constructs containing a matrix sequence may be used. Also, promote promoters to increase expression levels. An enhancer element may be used with the promoter. However, enhancer is inserted It does not necessarily need to be located upstream of the obtained heterologous DNA. Further, for example, heat induction Promoter or The use of inducible promoters, such as metal inducible promoters, is also known in the art. Preferred in some cases.   It is not necessary that the pox vector encodes all pox proteins. insect In addition, infect poxvirus target cells and express heterologous proteins You only need to code the necessary parts. Sometimes this part is poxgeno The part corresponding to the part sufficient for infection and expression of the virus is called a part corresponding to the part.   The recombinant poxvirus of the present invention may optionally comprise β-galactosidase, CAT, Contains markers such as neomycin or methotrexate resistance, which To detect (or select) the target cells of the host. Those skilled in the art will use such markers. For various viral vectors in certain target host cells Non-cytotoxic vectors can be screened. For example, β-galactosider Gene (lacZ) encoding any of the recombinant pox Virus into the target host cell. And the production of β-galactosidase is measured. β-gal expression is virus It is an indicator of virus infectivity and gene expression.Immune response   Specific immune response to myelin protein or its T cell-inducing epitope The answer is about 10^Five-10⁹Administer pfu of the recombinant poxvirus of the invention to a host Can be triggered by More preferably, about 10⁷-10⁹Use pfu However, this amount may vary depending on a number of factors, for example, the particular host used. You. Preferred hosts are domestic animals, captive animals or mammals such as humans. In some cases, the presentation is “boosted” by administering a booster antigen to the host. Is desirable. This administration may be after one to three months. Also, preferably the first booth At least one second "boost" may be administered 1 to 3 months after the injection. Mie The phosphoprotein and its T cell-inducing epitope share the same poxvirus vector Or more preferably, the antigen is antigenically related Using a second poxvirus vector derived from a non-poxvirus Alternatively, the antigen may be adjuvanted, e.g., in a pharmaceutically acceptable carrier. It may be administered directly using bunts or liposomes. IL-2, IL-6, Like IL-12 Various cytokines may be used as biological adjuvants, It can be administered systemically. Alternatively, such as cytokines or B7.1, B7.2 Gene into a recombinant pox vector And these molecules may be co-administered.   Adjuvants are, for example, RIBI Detox (Ribi immunoch) electrical), QS21 and incomplete Freund's adjuvant. Dosing Methods for preparing posomes are known.T cells   Epitope (s) of myelin protein or T cell induction thereof T cells that respond to a sexual epitope are obtained from peripheral blood mononuclear cells (PBMC) by standard methods. Obtained by For example, a lymphocyte separation medium gradient (Lymphocyte S) medium Medium gradient) (Organon T eknika Durham NC, USA) using the previously described procedure ( Boyum et al., Scand. Clin Lab Invest 21:77 -80 (1968)) to separate the PBMC. Washing For example, 10% human AB serum (Pel-Freeze Clin) ical System, Brown Dear, WI, USA). Lutamine, 100 U / ml penicillin and 100 μg / ml streptomycin Complete like RPMI 1640 (GIBCO) supplemented with Shin (GIBCO) Resuspend in medium. For example, about 2 × 10 5 in a 100 μl volume of complete medium⁶cell Of PBMC at a concentration of 96 wells in a flat bottom assay plate (Costar, Cam) (Bridge, MA, USA). Selected MBP peptide Was added to the culture at a final concentration of about 50 μg / ml and 5% CO_TwoIncubate for 5 days at 37 ° C. in a humid atmosphere containing MBP or The cytotoxic T cell-inducing epitope is removed from the medium and 5 days later a new human IL-2 (10 U / ml) is added to the culture and IL-2 containing medium is replenished every 3 days You. On day 16, the primary culture is restimulated with the same peptide (50 μg / ml). Release 5 × 10 with radiation irradiation (4,000 rad)⁶About 50μl of the same original PBMC And added as antigen presenting cells (APC) in complete medium. About 5 days later, same as above Human IL-2 The containing medium is added to the culture. Cells are restimulated for 5 days at 16 day intervals.Epitope mapping   T cells prepared according to the procedures described herein can be transformed into T cells such as cytotoxic T cells. (One or more of the myelin proteins or fragments thereof Number) of epitopes can be used. T cell induction of myelin protein One method of preparing the onset epitope is, for example, papain, trypsin or chymoto Lipsin (SIGMA Chemical Co. St. Louis, Mo. Limited protein digestion of proteins by enzymes such as Is the way. Alternatively, a fragment of the myelin protein can be A conventional person such as ifid Solid-Phase Technique It may be chemically synthesized by a method. For example, plating cytotoxic T cells, Different myelin protein fragments are added to different wells. At least one Specifically recognizes (eg, binds) a peptide fragment with two epitopes This facilitates the increase since only cytotoxic T cells continue to expand. Can be identified.   T cell-inducing epitope of myelin protein can substitute for complete protein . In addition, other fragments containing epitopes that enhance the ability to elicit a T cell response Can also be prepared.Mutants of myelin protein and DNA   Variants of myelin protein and DNA are included in the spirit and scope of the present invention. . For example, a degenerate variant of the MBP DNA sequence may have at least one degenerate variant. To encode a MBP amino acid sequence that contains nucleotide changes and consequently Equal to the MBP sequence except that one or more alternative codons are used No. There are 61 codons in 20 common amino acids, and many amino acids have more than one It will be understood by those skilled in the art that they are encoded by (optional) codons (Darn ell et al., eds., Scientific American Books, In. c. , 1986).   Additional variants are included within the spirit and scope of the present invention. For example, the recombination of the present invention Poxviruses contain at least one nucleotide change and consequently The MBP amino acid sequence One or more alternative codons are used to encode The alternative codon is a conservative amino acid, that is, another amino acid because it has similar properties. Includes variants that are equivalent to the MBP DNA sequence except for those that code for substitutable amino acids Include. Examples of conservative amino acid substitutions include valine, lysine, and glycine Arginine, leucine for valine, serine for threonine, etc. What is it? Of course, a conservative amino acid substitution may be Should not substantially affect the tope.   In yet another example of a mutant, the recombinant poxvirus of the invention has at least one Contains three nucleotide changes and consequently encodes the MBP amino acid sequence. One or more alternative codons are used to Is different from the corresponding amino acid in the MBP amino acid sequence. A mutation equal to the MBP DNA sequence except that it encodes an amino acid with the property value It also includes the body. In this case, a non-conservative amino acid substitution is made at the MBP (one or more ) Substantially does not affect T cell-induced epitopes.   Alternatively or additionally, the recombinant poxvirus may have deletions and / or insertions. Do not substantially affect the T cell-inducing epitope (s) of MBP. The deletion of the MBP DNA sequence or the deletion of the MBP DNA sequence. The insertion of a foreign gene.   The T cell-inducing epitope of MBP in polypeptide fragments is another type MBP mutant.Therapeutic composition   The recombinant poxvirus of the present invention is, for example, a trauma method, transdermal, subcutaneous, intramuscular, Administer to the appropriate host using any acceptable route, such as intravenous or intraperitoneal injection. Can be given.   The therapeutic compositions of the present invention preferably comprise one or more recombinant polypeptides of the present invention. Virus, and is typically pharmaceutically acceptable, such as saline. Aqueous or non-aqueous sterile solution, suspension or emulsion in combination with a carrier It is administered to a suitable host in a single dosage form. Parenteral dosage forms may also include excipients. Polyalkylene glycols such as commonly used polyethylene glycol Crude oil, hydrogenated naphthalene, etc. (For an overview, see Remington's Pharmaceutical ical Sciences (Mack Pub. Co., Easton, P. A, 1980).   The therapeutic composition of the present invention may be used as the sole active ingredient in a medicament, Is used in combination with other compounds and / or therapeutics to help treat inflammatory desheathing disease Is also good.   The amount of recombinant poxvirus in the therapeutic composition depends on the amount of virus administered, It can vary depending on many factors, such as the toxicity of the virus selected, the route of administration, and the like. one Generally, for parenteral administration, the compositions should be prepared in a saline buffer of about 10%.^Five-10⁹p It is administered as an aqueous solution of fu. When the host is human, a typical dosage range is about 1 0^Five-10⁹recombinant avipox virus or suipox wis in the pfu range And preferably fowlpox or swinepox. However, the preference for therapeutic compositions The dosage depends on the type and degree of inflammatory desheathing disease, the general health of the patient, Toxicity and biological efficacy of specific recombinant poxviruses identified, composition excipients It depends on variables such as the composition of the agent and its route of administration.                               Example 1                           Vector construction Pox virus   Many poxviruses are live viral vectors for the expression of heterologous proteins (Cepko et al., (1984): Marin et al., (1987); Lo we et al., (1987); Panicali & Paoletti (1982). ); Macett et al., (1982)). Typical chicken pox, pig pork Virus and vaccinia have accession numbers VR-229, VR-363 and And VR-325 from ATCC. DNA vector for recombination with parent virus in vivo   The gene encoding MBP or its T cell-inducing epitope is Virus gene, such as chicken pox virus, swine virus, or virus In addition to the normal expression of the parental viral protein, Insert so that expression by ills is also possible. This is a living organism with poxvirus Achieved by first constructing a DNA donor plasmid for internal recombination Is done.   Generally, a DNA donor plasmid contains the following elements: (I) Prokaryotic origin of replication, which allows the vector to be amplified in a prokaryotic host. Becomes possible; (Ii) encoding a marker for selecting prokaryotic host cells containing the vector A gene (eg, a gene encoding an antibiotic resistance trait); (Iii) a desired plasmid in contact with a promoter capable of expressing the gene; At least one gene encoding an erin protein (ie, MBP); (Iv) parent flanking a construction element (iii) into which a foreign gene is inserted DNA sequence homologous to the region of the irs gene.   Donor plasmid for introducing multiple foreign genes into poxvirus The construction method is described in WO91 / 19803, the technology of which is incorporated herein by reference. Has been captured by Generally, a fragment containing a transcription promoter and a foreign gene Donor vector containing a fragment containing a homologous sequence to the parental viral gene DNA fragments for constructing E. coli are genomic DNA or cloned DN It can be obtained from the A fragment. Donna -Plasmids are monovalent, divalent or multivalent (ie, an inserted foreign gene Can contain one or more columns).   To identify a recombinant virus having foreign DNA inserted into the donor plasmid It is preferable that another gene encoding a marker is included. Like this Recombinant virus can be identified and isolated by using multiple marker genes. it can. Such marker genes may have antibiotic or chemical resistance traits. Genes (eg, Spyropoulos et al., (1988); Falk ner and Moss (1988); see Franke et al., (1985)), colon There is a gene such as the lacZ gene of the fungus, which allows the It is possible to identify colonies of the recombinant virus (Panicali et al., ( 1986)). Transfer of heterologous DNA into poxvirus   Homologous recombination between donor plasmid DNA and viral DNA in infected cells When this occurs, a recombinant virus incorporating the desired elements is formed. In vivo group Host cells suitable for recombination are generally capable of infecting viruses and plasmid vectors. Eukaryotic cells that can be transfected. Pox virus benefits Examples of suitable host cells for use include chicken embryo fibroblasts, HuTK143 (human ) Cells and CV-1, BSC-40 (both monkey kidney) cells. Pox wi Infection of cells and transfection of these cells by plasmid vectors The implementation is performed using techniques standard in the art (Panicali and Paule). tti, U.S. Pat. No. 4,603,112, WO 89/03429, both of which are incorporated herein by reference. Is incorporated by reference into the book)   After in vivo recombination, the recombinant virus can be Offspring can be identified. For example, a DNA donor vector may be It is designed to insert one or more foreign genes into the midin kinase (TK) gene. When measured, the virus containing the inserted DNA was TK^-And use this to sort (Macckett et al., (1982)). Or, if the marker Or co-transferring the indicator gene together with the foreign gene in question in the same manner as described above. It can be used to identify recombinant progeny. One preferred indicator gene is E. coli la. The recombinant virus expressing β-galactosidase is the cZ gene; Screening can be performed using the chromogenic substrate of the enzyme (Panicall et al., (19 86)). Characterization of antigens expressed by recombinant poxvirus   Once the recombinant poxvirus was identified, it was inserted using a variety of methods. The expression of the polypeptide encoded by the gene can be assayed. These people Black plaque assay (in situ enzyme immunoassay on viral plaque) ), Western blotting, radioimmunoprecipitation (RIPA) and There is an enzyme immunoassay (EIA). MBP or MBP-related protein, or Antibodies that recognize myelin proteins, such as the T-cell activation epitope of proteins, are commercially available. It can be prepared or prepared by a known technique. For example, these Antibodies against MBP, MBP-related proteins or their T-cell-derived epitopes Can be made by standard hybridoma techniques (Kohler et al., (1975). ); Kohler et al., (1976); Hammerling et al., Monoclo. nal Antibodies and T Cell Hybridomas , Elsevier NY, 1981; Harlow and Lane in.   Antibodies: A Laboratory Approach CS HSOB (1988); and Ausubel et al., Supra). Determination of antibody titer   Anti-MBP antibodies were quantified using ELISA before and 2 weeks after immunization, respectively. . For example, a microtiter plate was prepared using MBP (100 ng / well, SOUR CE), ovalbumin (100 ng / well, Sigma) or 1 × 10⁷ Coated with PBS containing V-Wyeth inactivated with PFU / well UV To run. Block the plate with 2% BSA in PBS, dry and use -20 ° C until storage. This plate was diluted 1: 5 serum and standard As a control, a monoclonal antibody against PSA (DAKO M750, Denm ark) for 24 hours at 4 ° C. 1% of this plate several times After washing with PBS containing BSA, horseradish peroxidase-labeled yeast Giant anti-human IgG or IgM heavy chain specific serum (1: 8000) (Souther nBiotechnology Associates, Birmingha m, AL) at 37 ° C. for 45 minutes, followed by HRP substrate system ( Kirkegaard & Perry Laboratories, Gait Hersberg, MD) according to the manufacturer's instructions. The absorbance at 405 nm of each well Bio-Tek EL310 microplate ELISA reader (Winoo ki, VT). Optimization of immune response to recombinant poxvirus   Administration of the recombinant poxvirus disclosed herein may be subject to It can provoke a strong and unwanted immune response to the virus. same Continuous boosting with repeated administration of the same vector will result in no virus from the host. It was rapidly cleared as needed and could not adequately express myelin protein temporally. May not be able to boost the immune response to That is, the recombinant pot of the present invention Multiple “boosts” using virus virus are possible, but any It is not always preferable to use the ruth repeatedly. Has antigenicity By utilizing recombinant poxviruses from different poxviruses, In many cases, the above problem can be solved.   According to the present invention, a high level immune response against a recombinant poxvirus is To minimize, for example, the first poxvirus (or Virus group) is chicken pox, the second and subsequent recombinant pox Xviruses are different genera such as orthopox, for example, vaccinia and Choose from Suipox.   Alternatively, another way to minimize unwanted immune responses is to have a suitable host range and / or Or to select an appropriate poxvirus with tissue specificity. For example , Other than animals whose primary host range used poxvirus in the gene delivery system You can choose from For example, if the host is not a permissive animal such as a human, Avipox like pox or swipox like pigpox can be used Wear. In the case of veterinary applications where the host is a pig, as in the examples, It is not advantageous to use the box. Therefore, if the host is out of host range, If poxurus is not available, an attenuated strain is desired. For example, Use highly attenuated strains of specific orthopox such as xinia (MVA strain) Or use traditional genetic or chemical mutagenesis techniques Further attenuated until further non-pathogenicity in normal host range It can also be converted.   The cellular properties of the poxvirus in question easily screened for infectivity and amplification efficiency One way to do this.                                 Example 1         Construction of recombinant chicken poxvirus expressing MBP   2.2 The gene encoding MBP was isolated from a human brain cDNA library kb cDNA fragment [Kamholz et al., PNAS (USA) 83: 4962-4966 (1986)]. Encodes all 516 nucleotides of MBP Sequence and 5 'untranslated region of 36 nucleotides as well as 3' untranslated region of 661 nucleotides A 1.2 kb DNA fragment containing the translation region was ligated to the chicken poxvirus plasmid vector. Inserted into the monitor. The resulting plasmid was named pt3064 and used Vaccinia virus 40K early / late promoter (Gritz et al., 199). 0) and the chicken poxvirus C1 Osumi bacteria controlled by a promoter (Jenkine et al., 1991) vT92 containing the lacZ gene was created. This plasmid was produced on March 2, 1996. Deposited with the ATCC on the 2nd and received the accession number 97491. The foreign gene is chicken Flanking the DNA sequence in the Bam HI J region of the virus genome (Jenk ins et al., 1991). POXV, a vaccine strain of chicken poxvirus AC-TC (Schering Corp .. ) Induced strain was used as parent virus. Recombinant chicken pox Ils was a chicken pox virus transfected with pT3064. In chicken embryo epithelial (CED) cells infected with Between the chicken poxvirus sequence in Nome and the corresponding sequence in pT3064 Achieved by performing homologous recombination. The recombinant virus named vT92 was luogal (Life Technologies; Gaithersburg) g, MD), proliferated and identified in CED cells in the presence of β-galactosidase chromogenic substrate And the plaque was purified. MBP gene in chicken pox virus genome The insertion was confirmed by amplifying the inserted sequence by polymerase chain reaction (PCR). I did. MBP expression was determined by Western blot analysis using an MBP-specific antiserum. I checked.   Construction of recombinant vaccinia virus expressing MBP   Gene encoding human MBP was isolated from a human brain cDNA library 2 . It was excised from a 2 kb cDNA fragment [21]. All 516 nucleotides of MBP Coding sequence and 36 nucleotides of 5 'untranslated region and 661 nucleosides Cido's 3 'untranslated A 1.2 kb DNA fragment containing the translation region was transferred to a vaccinia virus transfer vector. Inserted in the The resulting plasmid designated pt115 contains vaccinia Vaccinia flanked by DNA obtained from the Hind IIIM region of the genome MBP relics controlled by the viral early / late promoter [22] Contains a gene. These flanking sequences are necessary for growth in human cells. An important vaccinia K1L host range gene [23] is included. This plasmid Deposited with the ATCC on March 22, 1996, and received accession number 97490. New   York City Health (NYCBH) vaccinia strain to parent virus Was used to construct a recombinant vaccinia virus. The parent virus is vA It was named bT33 (parent). The virus was released on May 15, 1989 by ATC C and deposited under the accession number VR-2240. This parent virus has a functional K Because of the lack of the 1L gene, rabbit kidney RK13 cells can grow efficiently. [24].   Construction of recombinant vaccinia virus utilizes standard homologous recombination techniques as described And achieved. Briefly, the vaccinia window in the vAbT33 (parent) genome Infects the viral sequence and parental virus RK13 cells transfected with the pT115 vector Homologous recombination was performed with the reaction sequence. The RK13 cell line is publicly available ( ATCC: accession number CCL37). Recombinant vaccinia with MBP gene Ils was selected by growing on RK13 cells. Recombinant containing all MBP genes Southern blot hybridization in which one of vaccinia viruses was digested with a restriction enzyme. Isolated. This recombinant virus was named vT15 (MBP) .   animal   Marmosets (C. jacchus) are from the University of California, San Fra ncisco (UCSF) bred in primate colonies. The dynamics used in this test These are the guidelines of the UCSF Animal Research Committee and National Re search Council, on the breeding and use of laboratory animals at the Laboratory Animal Resources Institute Was handled in accordance with the Commission's guidelines. Biweekly, up to 2.5 ml of blood Collected from animals. Phlebotomy and immunization were performed under brief anesthesia (ketamine , 10 mg intramuscularly).   Western analysis of MBP protein expression   Polyclonal rabbit anti-guinea pig MBP R120 antibody MBP was detected in vT15 (MBP) infected cells by Western blot analysis using It was shown to be expressed. This polyclonal antibody binds to human MBP . The preparation of antibodies has been described [25]. Equine herpesvirus gH Method for homologous recombination of a recombinant vaccinia virus containing a gene And used as a control recombinant vaccinia virus. The resulting recombinant cotton Xinia virus was named vAbT249 (control).   Briefly, Western experiments were performed with the parental vaccinia virus (vAbT33) or Shows recombinant vaccinia virus (vT15 (MBP)) in 2% fetal bovine blood Infect BSC40 cells in Dulbecco's modified Eagle's medium containing Qingqi It was carried out. Infect infected cells with hypotonic lysis buffer (150 mM NaCl, 0.0 Dissolved in 5% EDTA, 10 mM KCl, 1 mM PMSF) and then sonicated Processed. Electrophoresis of cell lysate and medium on SDS-10% acrylamide gel did. Transfer proteins to nitrocellulose membrane, and transfer nitrocellulose membrane Was incubated at room temperature with an antibody specific for MBP (R120), and then washed. And goat anti-rabbit phosphatase labeling Secondary antibody (AP, Kirkegaard & Parry Laboratory) ies, Gaithersburg, MD) and incubate with the manufacturer Color was developed according to the instructions.   Vaccination with recombinant vaccinia virus   Eight marmosets have vAbT249 (control) or vT15 (MBP) About 10⁷Pfu was vaccinated with four subcutaneous injections on the back. animal In some cases, 10 weeks after the first injection⁹pfu booster inoculation Was. In a monkey, a common bovine pot was obtained 10-14 days after the first vaccination. Crohn's disease symptoms and transient fever were observed. No other side effects were observed during this period Was not. About the pathological and immunological effects of vaccination on animals Pathological symptoms, such as the appearance and severity of skin disorders, anti-vaccinia T-cell activity and The antibody response described in detail below was measured and evaluated.   EAE induction in marmosets   Vaccination with vAbT249 (control) or vT15 (MBP) 21 to 35 days after, all animals were treated with 100 mg human total WM at 3 mg / ml Complete with H37Ra Immunization was performed with all Freund's adjuvant. 48 hours after immunization 10 for animals^TenKilled Bordetella pertussis (Lede) rr laboratories, NY) was administered intravenously. Daily mar Monitor the onset and progression of EAE by clinically observing mosets based on standard pathological criteria (See Massacesi, L., et al. (1995)). Animals at the end of the experiment Was anesthetized with a ketamine anesthetic and then a lethal dose of pentobarbital was administered intravenously ( (20 mg / kg) and then whole blood was collected.   Recombinant vaccinia virus expressing MBP in cells to PBMC infection   For MBP expression, vT15 (MBP) or VAbT249 (control) B-lymphoblast cells infected with Epstein-Barr virus and transformed with Epstein-Barr virus The strain was monitored in vitro using a strain (B-LCL). For the expression of MBP, PBMC were also monitored in vivo every 2-3 weeks after vaccination. Sa Ito spin samples (Shandon) were fixed in 50% ethanol and 1% bovine serum R120 rabbit polyclonal block with albumin phosphate buffered saline Guinea pig Stained with MBP antibody (1: 200), washed slides and labeled with peroxidase Incubated with anti-rabbit IgG antibody (Sigma). Color is 3-amino Run with -9-ethyl-carbazole (Sigma) and slide slides in hematoxylin / Double staining with eosin.   Briefly, expression of MBP in B-LCL cells was performed using the following method. Monkey autologous B lymphoblastoid cell line (BLCL) was isolated from freshly isolated 1 × 10^Five L-glutamine containing PBMC, gentamicin, and 10% FCS (Bio fluids, Rockville, MD) with 100 ml of RPMI1 At 640, 100 ml of S594 cells containing baboon Herpes Papio (D r. M. D. Miller, Harvard Medical School, New England Regional Primate Research h Center, Southborough, MA) 96 flat bottom plates (Costar, Cambridge, Mass.) Established in the well. After transformation, the cells were expanded and the medium was changed weekly.   We found that recombinant vaccinia virus vT15 It was discovered that myelin protein was expressed in PBMCs obtained from sown animals. This expression was paired with marmosets vaccinated with vAbT249 (control). It was not seen in the Teru group. During the period when MBP is expressed in each animal, There was a range of 15 to 45 days after vaccination.   Immune response to MBP and vaccinia antigen   T-cell reactions were performed on newly isolated 10 wells in 96-well round bottom plates.^FivePB To MC^ThreeStandard 72 hour growth assay using [H] -thymidine incorporation Investigation was carried out by the method [13]. Briefly, each plate is 200 μl / AIM Includes V (Gibco-BRL) and one of the following: nothing added (control); M BP 50 μg / ml; Proteolipid protein (PLP) 10 μg / ml; PH A 2.5 μg / ml; WM 0.1% (weight / volume); NYCBH Waxini A strain 2 μg / ml. Stimulation index is the ratio of unstimulated PBMC to stimulated PBMC Calculated as The term "stimulation index" refers to the recombinant vaccinia of the present invention. Recombination of the invention for the amount of T-cell activity in PBMC not exposed to ils T-cells in PBMC detected in biological samples exposed to body vaccinia virus Mean the ratio of the amount of activity are doing. Animals are sacrificed by anesthesia; spleen, lymph nodes are collected and then T-cells Proliferation assays were performed.   The production of MBP and vaccinia virus-conjugated antibodies is described in the manufacturer's instruction manual (Bio rad) and observed every 14-21 days using a dot blot filtration device [ 13]. Briefly, 250 ng MBP or 500 ng NYCB The H strain was adsorbed on a 0.45 μm nitrocellulose filtration membrane (Biorad) and Locked, a) serially diluted marmoset serum; b) peroxidase Labeled monkey IgG (Sigma, 1: 4,000 dilution); c) chromogenic substrate And diaminobenzidine (Pierce).   vT15 (MBP) recombinant vaccinia virus alleviates EAE.   In control animals vaccinated with vAbT249 (control), after immunization with WM Approximately 14-18 days, EAE developed (Table 1 and FIG. 1A). [12,13,26]. Control animals that did not develop EAE as clinical symptoms , But died from anesthesia 14 days after immunization. Table 1 is shown below You:  Recombinant vaccinia virus expressing MBP (vT15 (MBP)) In animals vaccinated with EAE, the appearance of clinical symptoms of EAE was significantly delayed (Table 1). And FIG. 1B). For example, two animals (animal numbers 344-92 and 353-92) have EAE For 63 days.   Clinical and pathological signs of EAE seen in animals vaccinated with vT15 Ensures delay or suppression compared to control animals vaccinated with vAbT249- Was done. adjuvant / bandetella pertussis WM In experiments before immunizing more than 30 animals with, more than 75% of the monkeys had 14- The disease developed within 28 days, and all cases became ill 60 days after immunization. That is, Onset of EAE (37-120 days) in vT15 (MBP) This is not likely due to the individual susceptibility of the animals in the inoculated group. Data is The preventive effect of vT15 (MBP) on vaccination is transmitted through the expression of MBP Nonspecificity of the immune response due to exposure to the vaccine virus It is not due to a typical modification.   One control animal vaccinated for neuropathology (animal number 346-92) at the time of acute EAE onset (22 days after immunization, 5 days after EAE onset) Eye, see FIG. 1A). Two more animals vaccinated with vT15 (MBP) First exemption for animals (animal numbers 344-92 and 353-92, see FIG. 1B) Checked on day 63. In control animals, typical C. acute E by Jacchus AE pathology was seen [12,13]. For example, simply at multiple places in the white matter of the CNS Nuclear cells and macrophages infiltrated around the blood vessels. Notable for such phenomena With severe coaxial desheathing and early gliosis. Abnormality was recognized in gray matter structure Was not. These disease findings were vaccinated with the control vaccinia virus. In the case of WM with a conventional adjuvant showing no improvement in EAE Untreated C. infected Same as the report for Jacchus. On the contrary When two animals vaccinated with vT15 were examined 63 days after immunization, perivascular The infiltration into the part was found to be slight, and was not accompanied by desheathing (FIG. 1, Table 1). . Thus, according to clinical and neuropathological criteria, recombinant MBP-vaccinia Prophylactic vaccination by C. protects the Jacchus marmoset from EAE Was.   Vaccinia virus despite being an FDA approved vaccine component May have encephalomyelitis pathogenic properties (Paoletti et al. (1993) ). For these reasons, we have developed a vaccine with the NYCBH strain of vaccinia virus. The inoculation itself is C.I. Jacchus was examined for increased EAE.   vAbT249 (control) or vT15 (MBP) recombinant vaccinia Marmosets in each group vaccinated with ils and injected with white matter (WM) Two animals were followed up for 163 days after WM injection (Table 1 and FIG. 1A B). Both groups show severe clinical symptoms (more than 3 degrees) and die or Was euthanized (animal number 91-92 (day 70); 94-92,3). 26-91 and 499-92 (each on day 120). Large shedding in control animals Extensive infiltration with dissemination was observed in white and gray matter (Table 1). Only However, in two animals vaccinated with vT18 (MBP), such clinical signs No condition was observed prior to sacrifice of the animals (FIG. 1B). These observations The result is that acute disseminated encephalomyelitis follows vaccination with vaccinia virus. Suggests to get up. Reduced or more attenuated vaccini Or use another poxve such as Avipox or Suipox. It is anticipated that these symptoms can be avoided by the use of a physician.Immune response to myelin and vaccinia antigens 1. T-cell reaction   MBP, proteolipid protein (PLP), and vaccinia virus (N YCBH) as a response to a stimulus^Three[H] -Thymidine incorporation, vaccination PBMC was measured continuously from the start date to the day of death. The results are summarized in FIG. Was.   T-cell activity against MBP: Two animals vaccinated with vT15 (MBP) In marmosets, transient and mild (ie, stimulation index 2) prior to induction of EAE by WM ) Were observed (animal numbers 344-92 and 353-92, FIGS. 2A and 2C). reference). This result indicates that vaccination with recombinant vaccinia virus This indicates that the immune system against the antigen appears in the living body. Active immunization with WM And T-cell proliferative response to MBP vaccinated with vAbT249 (control) Up to 15 and 22 days in the two animals and up to 62 days in the third control monkey. I was guessed. However, vaccination with vT15 (MAB) T cell proliferation to MBP did not occur until 74 days after immunization. This group In one of the animals (animal numbers: 91-92), T -No cell response was observed. T-cell reactivity to PLP in these experiments Was not shown.   T-cell reactivity to vaccinia virus: Vaccination or WM T-cells against NYCBH in both experimental and control groups after immunization with A proliferative response was observed (see FIGS. 2C and D). This is because of the recombination Subcutaneous injection of the human vaccinia virus Effective for Jacchus cell-mediated immune response This indicates that the stimulus has been effectively obtained. Later in animals that developed acute disseminated encephalomyelitis Increased the anti-vaccinia T-cell response.   T-cell reactivity to PLP: Against PLP in animals immunized with WM No examples showed T-cell reactivity. However, one animal that developed EAE was strong. Proliferative response to low PLP. Antibody reaction   A) Antibody to MBP: Serum antibody titer is bT15 (MBP) Or vAbT249 (control) recombinant vaccinia virus Was not detected after vaccination. However, all animals were immunized with WM 14-21 days later, antibodies binding to MBP appeared.   b) Antibody to vaccinia virus: against the NYCBH strain of vaccinia virus Antibodies were detected 14-21 days after the first vaccination. This result Humoral immunity against vaccinia antigen as well as T-cell proliferation reactivity Indicates that it was induced by   Part of the antigen presentation by the vaccinia virus vector is MHC class I antigen Both occur via the endogenous antigen presentation pathway. This allows for regulatory T-cells, especially The proliferation of CD8 + T- cells is stimulated. I do not intend to tie it to a specific theory The reduction in EAE observed in these experiments indicates that certain T-cell subclasses Suppression or secretion of inhibitory cytokines from regulatory T-cells Or both. Such defense mechanisms (or other Mechanism) is thought to be invalidated by the pathogenicity of vaccinia virus encephalomyelitis You.   Therefore, a recombinant poxvirus with reduced encephalomyelitis pathogenicity A recombinant chicken poxvirus as disclosed above. It is used in place of vaccinia virus in mammals, especially livestock, captive animals, and humans. Elimination or delay of the onset of inflammatory desheathing disease.   Some of the above components and methods are suitable for clinical applications or veterinary applications. Can be used as Such kits can be used as pharmaceuticals The recombinant poxvirus of the present invention or the recombinant pox virus of the present invention may be contained in a carrier. It contains one or more cells or homogeneous populations of cells that contain ills. In particular, cells or The homogenous population of cells is the host that is or is suspected of having the inflammatory desheathing disease described in this document. Myelin protein capable of binding to T-cells derived mainly, preferably MBP, MBP- Cells expressing intracellular related proteins or their T-cell activating epitopes Alternatively, it can be used as part of a diagnostic kit as a homogeneous population of cells. T-cells and this book The binding between the disclosed cells or homologous cells can be achieved by using ordinary immunization techniques. Can be detected.   All publications cited herein are subject to ordinary skill in the art to which this invention pertains. Is shown. All publications incorporated by reference in this document are quoted The specific range as well as the specific description is incorporated in its entirety.   The invention is described in some detail by means of figures and examples in order to make the understanding more clear. Described in detail, but without departing from the spirit and scope of the appended claims. It will be easy to see that changes and modifications can be made.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/275 A61K 39/275 48/00 48/00 C12N 5/10 C12N 7/00 7/00 5 / 00 B (71) Applicant The Regions of the University of California, California, CA 94612-3550, Auckland, Lakeside Drive 300, Twenty Second Floor (72) Inventor Letvin, Norman United States, Massachusetts 02158, Newton, Brackett Road 36 (72) Inventor Jenine, Claude P. United States of America, California 94612-3550, Auckland, Lakeside Drive 300, Twenty Cand Floor (72) Inventor Grits, Linda United States, Massachusetts 02143, Somerville, Emerson Street 3 (72) Inventor Hauser, Steven H. United States of America, California 94612-3550, Oakland, Lakeside Drive 300, Twenty Second Floor (72) Inventor Panicali, Dennis USA, Massachusetts 01720, Acton, Non-Set Pass 114

Claims (1)

[Claims] 1. An operably linked heterologous DNA sequence encoding a myelin protein A recombinant poxvirus having at least one insertion site comprising Wherein the recombinant poxvirus is capable of regulating the host immune system. A recombinant poxvirus derived from a poxvirus. 2. A heterologous DNA sequence encoding MBP or a variant thereof. The recombinant poxvirus according to claim 1. 3. Poxvirus is orthopoxvirus, avipoxvirus or 2. The recombinant pock according to claim 1, wherein is a suipox virus. Virus. 4. Poxvirus selected from the group consisting of vaccinia, fowlpox or swinepox The recombinant poxvirus according to claim 3, which is selected. 5. Characterized in that the poxvirus is wild-type or attenuated The recombinant poxvirus according to claim 1. 6. 2. The recombinant pot according to claim 1, wherein the immune response is a T cell response. Kusu virus. 7. Cells express myelin protein and elicit a cytotoxic T cell response A cell infected with the poxvirus according to claim 1, characterized in that: 8. The recombinant poxvirus of claim 1 in a pharmaceutically acceptable carrier. A therapeutic composition for reducing or prolonging the onset of inflammatory desheathing disease, comprising: . 9. Poxvirus selected from the group consisting of fowlpox, vaccinia and swinepox 9. The therapeutic composition according to claim 8, wherein the composition is used. 10. The myelin protein is MBP or a variant thereof. A therapeutic composition according to claim 8. 11. A method for generating an immune response to a myelin protein, comprising: (A) a sufficient amount of the first recombinant plasmid to present the myelin protein to the immune system; Introducing a virus into a host; (B) expressing the myelin protein in a cell; (C) presenting the myelin protein to the immune system;   The first recombinant poxvirus encodes myelin protein At least one operably linked DNA segment comprising A method comprising having an insertion site. 12. In addition, at least one routine after introduction of the first recombinant poxvirus At an interval, additional myelin protein or its cytotoxic T cell-inducing properties 12. The method of claim 11, comprising contacting the epitope with a host. Method. 13. Operatively linked to a DNA segment encoding a myelin protein Recombinant poxvirus having at least one insertion site including in a closed state To bring additional myelin protein into contact with the host by introducing 13. The method according to claim 12, wherein the method comprises: 14． The myelin protein is MBP or a variant thereof. 14. The method according to claim 13. 15. A method of presenting a myelin protein to a host immune system, (A) sufficient amount of myelin protein or its cytotoxic T cell-induced epithelium Contacting the host with the host; (B) at least one subsequent regular interval of additional myelin protein Contacting the quality with the host.   Contacting the additional myelin protein with the host comprises At least one containing an encoding DNA segment in operably linked state Performed by introducing a first recombinant poxvirus having an insertion site A method characterized by the following. 16. The first recombinant poxvirus is Suipox virus, Avipox Glue consisting of virus, capripox virus and orthopox virus 16. The method of claim 15, wherein the method is derived from a virus selected from the group consisting of: . 17． Characterized in that the poxvirus is vaccinia, fowlpox or swinepox The method of claim 16. 18. The first recombinant poxvirus is derived from avipox and the second recombinant poxvirus is Virus is suipox virus, capripox virus and orthopox virus Pox virus selected from the group of pox viruses 17. The method according to claim 13 or 16, wherein the method is derived from irs. 19. First or second recombinant poxvirus derived from attenuated poxvirus The method of claim 18, wherein: 20. Wherein the recombinant poxvirus exhibits low replication efficiency in the host. The method according to claim 11 or 15, wherein 21. The recombinant according to claim 1, which is vT92 or vT15. vector.