WO1997035997A2 - Poxvirus recombinant codant pour la proteine de la myeline et destine a un usage therapeutique - Google Patents

Poxvirus recombinant codant pour la proteine de la myeline et destine a un usage therapeutique Download PDF

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WO1997035997A2
WO1997035997A2 PCT/US1997/005217 US9705217W WO9735997A2 WO 1997035997 A2 WO1997035997 A2 WO 1997035997A2 US 9705217 W US9705217 W US 9705217W WO 9735997 A2 WO9735997 A2 WO 9735997A2
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virus
recombinant
pox virus
pox
myelin protein
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PCT/US1997/005217
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WO1997035997A9 (fr
WO1997035997A3 (fr
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Norman Letvin
Claude P. Genain
Linda Gritz
Stephen H. Hauser
Dennis Panicali
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Therion Biologics Corporation
Beth Israel Deaconnes Medical Center
The Regents Of The University Of California
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Priority to JP9534670A priority Critical patent/JP2000507824A/ja
Priority to AU26586/97A priority patent/AU2658697A/en
Publication of WO1997035997A2 publication Critical patent/WO1997035997A2/fr
Publication of WO1997035997A3 publication Critical patent/WO1997035997A3/fr
Publication of WO1997035997A9 publication Critical patent/WO1997035997A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Inflammatory demyelinating diseases of the central nervous system such as multiple sclerosis (MS) and Guillian-Barre syndrome are believed to involve an immune response against CNS autoantigens.
  • CNS central nervous system
  • EAE experimental allergic encephalomyelitis
  • MBP myelin basic protein
  • MBP-reactive T-cells can typically be recovered from fluids of MS patients [1 , 2 and 3]. These T-cells are concentrated in the cerebrospinal fluid (CSF) of MS patients when compared to controls with other neurological diseases (2).
  • CSF cerebrospinal fluid
  • MBP-reactive T cells have also been directly detected in MS lesions (3).
  • MBP is a major antigen since the disease can be induced either by active immunization with MBP, antigenic fragments of MBP, or by adoptive transfer of MBP- reactive T cells (4,5).
  • MBP is one component of myelin protein.
  • the MBP gene encodes several MBP-related proteins through alternative RNA splicing (see Lemki, G. (1988), Mikoshiba, K., et al. ( 1 991 ), Kamholz, J.,
  • Animals that are susceptible to EAE are recognized models for the study of human inflammatory demyelinating diseases such as, e.g.,
  • EAE can typically be induced by immunization with whole myelin, MBP, antigenic fragments of MBP, or by adoptive transfer of MBP- reactive T-cells [ 1 2, 1 3, 4, 5].
  • Rodents with EAE generally recover from the disease, indicating that the immune system suppresses the disease, e.g., by providing cytotoxic T-cells or anti-inflammatory cytokines [6-1 1 ].
  • Human MS however, is a relapsing and remitting disease which differs from most forms of acute rodent EAE. Little is known about the factors responsible for relapses, and the role of regulatory T-cells (e.g., T suppressor cells) in the human disease has not been thoroughly investigated.
  • EAE can be induced by active immunization with whole white matter (WM), MBP, or by adoptive transfer of MBP-reactive-T- cells or clones [12- 1 ].
  • the marmoset is an especially advantageous model for the study of inflammatory demyelinating disease, in part because siblings share a bone marrow chimerism which permits adoptive transfer of lymphocytes between genetically distinct individuals.
  • T-cell and antibody reactivity against MBP is detected at the onset of disease and can be observed in peripheral blood mononuclear cell (PBMC) preparations ( 13).
  • PBMC peripheral blood mononuclear cell
  • Pox viruses of the family Poxviridae (pox viruses) are useful as vectors for the delivery of foreign genes and gene products in many clinical and research settings.
  • Pox viruses of the genus Orthopoxvirus, particularly vaccinia are used for several reasons. Among these are: (a) its wide use in humans in the eradication of smallpox; (b) its ability to infect a wide range of cells, including professional antigen presenting cells, and express the inserted gene product (i.e. foreign gene product) in a manner that has the potential to be processed in the context of class I and/or class II MHC molecules; and (c) use as a recombinant vaccine in the treatment of certain tumors (Kantor, J. et al. (1992)).
  • pox viruses have been useful as vectors for vaccines and the expression of foreign genes in cells, such as viruses of the genus Avipoxvirus such as, e.g., fowl pox, canary pox and viruses of the genus suipox such as swine pox.
  • a recombinant pox virus to intracellularly express a myelin protein or a T-cell eliciting epitope thereof, we can ameliorate or delay the symptoms of an inflammatory demyelinating disease in a vertebrate host.
  • the host immune system will thereafter substantially tolerate the myelin protein, thereby helping to ameliorate or delay the symptoms of the disease.
  • intracelluler expression we mean that the process of transcription and translation of a protein occurs within the host cell. The protein, itself, may then be maintained in the cytoplasm, or transported to the nucleus, the cell membrane, other areas within the cell or into the extracellular space.
  • the recombinant pox virus has at least one insertion site containing a DNA segment encoding the myelin protein or a T-cell eliciting epitope thereof, which DNA segment is operably linked to a promoter capable of expression in the host.
  • myelin protein is meant those proteins which constitute the concentric oligodendrocyte wrapping around axons such as, e.g., MBP, PLP, MOP and MAG.
  • a preferred myelin protein is MBP, MBP-related proteins, and T-cell eliciting epitopes thereof.
  • Other proteins include MOP and MDP (myelodendritic protein).
  • a preferred method of the present invention comprises introducing a sufficient amount of a first recombinant pox virus vector into a host to stimulate an immune response, and present the myelin protein to the immune systems.
  • a "boost” Preferably, one uses a first recombinant pox virus derived from a pox virus of a different genus for the "boost", e.g., first administer using a recombinant pox virus derived from an avipox, then boost with another derived from suipox.
  • the first recombinant pox virus vector comprises a DNA segment which encodes the myelin protein or T-cell eliciting portion thereof.
  • the additional antigen as aforesaid may be added, e.g., by using a second recombinant pox virus from an immunologically distinct pox virus, typically from a different genus than the first pox virus.
  • additional myelin protein or T-cell eliciting fragment thereof may be added by contacting the host with the myelin protein formulated with an adjuvant or in a liposomal formulation or contacting the host with DNA encoding the myelin protein either as direct DNA or in an alternative viral vector.
  • the present invention also features a cell, preferably a homogeneous population of cells, which includes either the recombinant pox virus DNA or the recombinant pox virus, where the encoded myelin protein is expressed intracellularly in a sufficient amount to elicit an immune response against the myelin protein.
  • the cell is preferably one which is capable of supporting virus or protein expression for at least 48 hours, such as, e.g., a mammalian, avian, reptilian, or insect cell line.
  • the present invention also features therapeutic compositions which include one or more recombinant pox virus disclosed herein where the virus(es) is provided in a pharmaceutically acceptable carrier.
  • the therapeutic compositions can be administered to a mammal, preferably a human, as a means for ameliorating or delaying the onset of an inflammatory demyelinating disease such as, e.g., MS.
  • the therapeutic compositions of the invention can be administered alone or as adjuncts to known therapies for treating inflammatory demyelinating disease such as, e.g, Beta-interferon, adrenocorticotropic hormone (ACTH) and corticosteroids for the treatment of MS.
  • therapies for treating inflammatory demyelinating disease such as, e.g, Beta-interferon, adrenocorticotropic hormone (ACTH) and corticosteroids for the treatment of MS.
  • therapies for treating inflammatory demyelinating disease such as, e.g, Beta-interferon, adrenocortic
  • inflammatory demyelinating disease in, for example, mammals and in a domesticated animal such as, e.g., a dog, cat, horse, rabbit, mouse, pig, cattle, reptile, gerbil, bird, sheep, goat and the like, or a captive animal such as a primate (e.g., chimpanzees, etc.) is within the scope of the present invention.
  • a domesticated animal such as, e.g., a dog, cat, horse, rabbit, mouse, pig, cattle, reptile, gerbil, bird, sheep, goat and the like
  • a captive animal such as a primate (e.g., chimpanzees, etc.)
  • inflammatory demyelinating disease means any immunological response which destroys, damages or removes the myelin sheath of nerve fibers.
  • An inflammatory demyelinating disease in a mammal, particularly a human, can be identified by such damage.
  • the invention also features a method for generating an immune response to a myelin protein in a host, the method including: a. contacting the host with a sufficient amount of the myelin protein or T-cell eliciting epitope thereof; and b. at least one periodic interval thereafter, contacting the host with additional myelin protein or T-cell eliciting epitope thereof.
  • the invention also features a method of manufacturing a myelin protein capable of eliciting an immune response.
  • the method includes the steps of: a) synthesizing a recombinant pox virus, preferably an avipox such as fowl pox, or canary pox, a capripox, or a suipox such as swine pox, which virus includes an operably linked DNA segment encoding a myelin protein, preferably MBP, MBP-related protein, or a T-cell eliciting epitope thereof; b) introducing the recombinant pox virus into a mammalian host, preferably a human, under conditions which permit the infection of cells which support gene expression by the virus, preferably dermal cells or muscle cells; and c) expressing and appropriately presenting the myelin protein intracellularly so that an immune response is elicited in the host.
  • a recombinant pox virus preferably an avipox such
  • the method is also useful for producing cells (or cell lysate thereof) which can be used to detect the onset of or evaluate the progression of an inflammatory demyelinating disease in a host.
  • the cells appropriately present the myelin protein and are therefore recognized by T-cells isolated from the host.
  • the killing by the T-cells can be detected by standard techniques such as, e.g., chromium release from cells labelled with radioactive chromium.
  • the method further comprises isolating the cells infected by the recombinant pox virus and using the cells, either as whole cells or cell fractions, as an immunogen for the production of antibodies, preferably monoclonal antibodies, which bind intracellularly expressed myelin protein in the infected cell.
  • antibodies preferably monoclonal antibodies, which bind intracellularly expressed myelin protein in the infected cell.
  • the antibody may also bind a T-cell eliciting epitope of the myelin protein.
  • such antibodies can readily be made by those skilled in the art.
  • the method further comprises isolating cells from the infected host (or isolating the infected cells themselves), and using them, either as whole cells or cell fractions, to induce or stimulate an immune response in a second host to ameliorate or delay the onset of an inflammatory demyelinating disease in a mammal such as, e.g., a human.
  • Figures 1 A and B shows two graphs which depict the clinical course of EAE in vAbT249 (control) and vT1 5 (MBP) vaccinated C. jacchus marmosets.
  • Figures 2 A, B, C and D shows four graphs which illustrate the proliferative responses in PBMC of WM-immunized C. jacchus marmosets vaccinated with vT15 (MBP) ( Figures 2 A and C) and vAbT249 (control) ( Figures 2B and D).
  • Figures 2A and B show in vitro stimulation with MBP (50 ⁇ g/ml).
  • Figures 2C and D show stimulation with NYCBH vaccinia antigen (2 ;g/ml).
  • Viruses of the family Poxviridae are well known cytoplasmic viruses.
  • genetic material expressed by recombinant pox viruses of the invention typically remains in the cytoplasm and does not have the potential for inadvertently integrating into host cell genes.
  • becai .se pox viruses have large genomes, they can readily be used to deliver a wide range of genetic material including multiple foreign genes (i.e., act as a multivalent vector).
  • Pox viruses disclosed herein are typically viruses of the family Poxviridae, preferably selected from the group consisting of avipox, capripox, orthopox, entomopox and suipox.
  • Preferred avipox viruses include fowl pox, canary pox, pigeon pox, turkey pox, quail pox.
  • An especially preferred avipox is fowl pox.
  • Preferred orthopox viruses include vaccinia, cowpox, mousepox (ectromelia), rabbitpox, racoon pox, and monkey pox.
  • An especially preferred orthopox is vaccinia.
  • the suipox is swine pox
  • the capripox is sheep or goat pox virus.
  • a pox virus whose host range is restricted and does not include humans.
  • a restriction can be natural such as by the use of avipox, capripox, entomopox or suipox in a human. Restriction can also be accomplished by attenuating (i.e. weakening) the pox virus whose host range is a human to a sufficient degree that pox virus will no be virulent. See for example, the NYVAC strain of vaccinia, U.S. Patent No. 5,494,807, herein incorporated by reference. See also the MVA strain in U.S. Pat. No. 5, 185, 146, herein incorporated by reference. The use of such attenuated pox viruses as vectors is particularly preferred in a host whose immune system is impaired.
  • viruses include those derived from herpes virus, polyoma viruses (e.g, SV40 and polyoma), adenoviruses, adeno associated viruses, as well as RNA viruses such as retroviruses and picornavirus such as polio virus, Sindbis, Venezuelan equine encephalitis.
  • viruses include iridoviruses such as frog virus, and African swine fever virus.
  • the recombinant pox virus exhibits a low replicative efficiency in the infected (i.e. target) cells of the host.
  • replicative efficiency can be determined by conventional virological and cell culture techniques such as, e.g., infecting with virus and then quantitating the amount of viral progeny produced by the infected cell by titration on permissive cells (e.g. by viral plaque assay). The amount of progeny virus so produced can be expressed over the total number of cells used in the experiment.
  • Preferred pox viruses used in accordance with the present invention preferably produce, on average, no more than about 10 productive (i.e., infectious) progeny per cell, more preferably, no more than about 1 productive progeny, still more preferably, no more than 0.1 progeny per cell.
  • a productive progeny is one that will infect and replicate in a permissive host cell.
  • the recombinant pox viruses made therefrom will not result in sustained replication and infection of other cells.
  • the vector and transformed cells will not adversely affect cells in the host animal at locations distant from where the target cell is.
  • more virulent pox virus can be used to make the recombinant pox viruses of the present invention, provided that the virulence of the virus has been reduced by, e.g., selection or chemical or genetic mutagenesis so as to produce an attenuated strain of the virulent pox virus. See previously incorporated U.S. Pat. No. 5,494,807.
  • the recombinant pox viral vector elicits a CD8 + response.
  • the myelin encoded antigen specifically triggers the down regulation of myelin antigen-specific CD4 cells, perhaps through a CD8 + cell mediated response.
  • the heterologous DNA sequence to be inserted into a pox virus can be placed into a donor plasmid, bacteriophage or virus such as, e.g., an E. coli plasmid such as pBR322 or pUC19.
  • a donor plasmid preferably includes an origin of replication (ORI), and one or more detectable markers such as, e.g., an antibiotic resistance gene (e.g., ampicillin or tetracycline) for propagation in E. coli.
  • ORI origin of replication
  • detectable markers such as, e.g., an antibiotic resistance gene (e.g., ampicillin or tetracycline) for propagation in E. coli.
  • the heterologous DNA is typically flanked by DNA sequences which are homologous to the section of pox virus DNA flanking the insertion site.
  • the heterologous DNA gene sequence to be inserted is ligated to a suitable promoter.
  • the promoter-gene linkage is then operably positioned in the plasmid so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA which is the desired insertion region.
  • a pox promoter is generally used.
  • the resulting recombinant plasmid is then used to transform E. coli where the cells are then propagated under selective conditions to reproduce the recombinant plasmid.
  • the reproduced plasmid is then purified by standard means.
  • other plasmids as well as a eukaryotic vector e.g., SV40 propagated in green monkey cells
  • may be employed to propagate the heterologous DNA See Sambrook et al. supra).
  • a preferred method of making a recombinant pox virus of the present invention is by inserting the heterologous DNA of the plasmid into a pox virus by genetic recombination.
  • the recombinant plasmid containing the heterologous DNA sequence to be inserted is transfected into a cell culture, e.g., chick embryo fibroblasts, along with the pox virus, e.g., fowl pox or swine pox.
  • the pox virus e.g., fowl pox or swine pox.
  • Recombination between homologous pox DNA in the plasmid and the viral genome respectively results in a recombinant poxvirus modified by the presence of the promoter-gene construct in its genome.
  • the site of recombination is one which does not affect virus viability.
  • virus viability is meant the capability of the recombinant pox virus to produce infectious viral particles within about 48 hours in a permissive host cell.
  • virus viability it may be desirable for the insertion to affect virus viability such as when the insertion attenuates the resulting recombinant pox virus. See discussion, infra. If it is not desirable to substantially affect virus viability, the artisan can readily identify such regions by, for example, by testing pre-determined segments of virus DNA for regions that support genetic recombination without seriously affecting virus viability.
  • TK thymidine kinase
  • insertion regions which do not substantially affect virus viability include, for example, the Hindlll M fragment.
  • insertion regions include, for example, the BamHl J fragment [Jenkins, et al., (1991 )] the EcoB ⁇ -Hind ⁇ fragment, EcoRV-HindW fragment, BamHl fragment and the HindlW fragment set forth in EPO Application No. 0 308 220 A1 . [Calvert, et al., (1993); Taylor, (1988); Spehner, et al., (1990) and Boursnell, et ai. (1990)].
  • preferred insertion sites include the thymidine kinase gene region.
  • a promoter operably linked to the heterologous DNA i.e., the heterologous DNA, promoter and pox virus are in a suitable spatial relationship which supports transcription of the heterologous DNA.
  • the promoter must be placed so that it is located upstream from the DNA to be expressed. Promoters are well known in the art and can be readily selected depending on the host and the cell type desired. For example in pox viruses, promoters derived from pox viral promoters are used.
  • Enhancer elements can also be used in combination with promoters to increase the level of expression, although enhancers need not always be located upstream of the inserted heterologous DNA.
  • inducible promoters such as, e.g., heat or metal inducible promoters, are also well known in the art and will be preferred in some instances.
  • pox vector encode all pox proteins.
  • a recombinant pox virus of the invention may optionally include a marker, such as /5-galactosidase, CAT, neomycin or methotrexate resistance, whereby the target cells of the host may be detected (or selected) .
  • a marker such as /5-galactosidase, CAT, neomycin or methotrexate resistance
  • the use of such a marker allows the skilled artisan to screen various viral vectors for those that are non-lytic or non-cytopathic in a particular target host cell.
  • the gene encoding ⁇ - galactosidase (lacZ) can be inserted into any recombinant pox virus disclosed herein, whereby the modified virus vector is then introduced into the target host cell and the production of / ⁇ -galactosidase is measured. Expression of / 5-gal provides an indication of viral infectivity and gene expression.
  • a specific immune response against a myelin protein or T-cell eliciting epitope thereof can be generated by administering between about 10 5 -10 9 pfu of a recombinant pox virus of the invention, to a host. More preferably about 10 7 -10 9 pfu is used, although this amount may vary depending several factors such as, e.g., the particular host used.
  • the preferred host is a mammal such as, e.g., a domesticated animal, a captive animal, or a human, in some instances, it is desirable to "boost" the presentation by administering additional antigen to the host. This may be one to three months later.
  • the myelin protein T-cell eliciting epitope thereof may be administered using the same pox virus vector or, more preferably the antigen is administered using a second pox virus vector from an antigenically unrelated pox virus, or alternatively, the antigen may be administered directly using, for example, an adjuvant or liposome in a pharmaceutically acceptable carrier.
  • Cytokines e.g., IL-2, IL-6, IL-12 may be used as biologic adjuvants and can be administered systemically to the host.
  • cytokines or co-stimulatory molecules e.g., B7.1 , B7.2, can be co-administered via co-insertion of the genes encoding the molecules into the recombinant pox vector.
  • Adjuvants include, for example, RIBI Detox (Ribi Immunochemical), QS21 and incomplete Freund's adjuvant. Methods for making liposomes for administration are known.
  • T-cells that react against the epitope(s) of myelin protein or a T- cell eliciting epitope thereof can be obtained from peripheral blood mononuclear cells (PBMC) by standard methods.
  • PBMC peripheral blood mononuclear cells
  • PBMC can be separated by using Lymphocyte Separation Medium gradient (Organon Teknika, Durham, NC, USA) as previously described [Boyum, et al., Scand J. C/in Lab Invest 21 : 77-80 (1968)].
  • Washed PBMC are resuspended in a complete medium, for example, RPMI 1640 (GIBCO) supplemented with 10% pool human AB serum (Pel-Freeze Clinical System, Brown Dear, WI, USA), 2mM glutamine, 100 U/ml penicillin and 100 /g/ml of streptomycin (GIBCO).
  • PBMC at a concentration of about 2 x 10 5 cells in complete medium in a volume of, for example, 1 00 ⁇ are added into each well of a 96-well flat-bottom assay plate
  • Protein antigen such as selected MBP peptides is then added into the cultures in a final concentration of about 50 ⁇ g/ml and incubated at 37°c in a humidified atmosphere containing 5% CO 2 for 5 days. After removal of the MBP or cytotoxic T-cell eliciting epitope thereof from the media, the cultures are provided with fresh human IL-2 ( 10U/ml) after 5 days and replenished with IL-2 containing medium every 3 days. Primary cultures are restimulated with the same peptide (50 //g/ml) on day 16.
  • T-cells prepared as described herein can be used to identify the epitope(s) of a myelin protein or fragment thereof, that are recognized by T-cells including cytotoxic T-cells.
  • One method of preparing a T-cell eliciting epitope of a myelin protein includes limited proteolytic digestion of the protein with enzymes such as, e.g., papain, trypsin, or chymotrypsin (supplied by SIGMA Chemical Co. St. Louis, Mo.).
  • fragments of a myelin protein can be chemically synthesized by conventional methods such as, e.g., the Merrifield Solid-Phase Technique.
  • cytotoxic T-cells can then be plated and the different myelin protein fragments added to different wells. Only cytotoxic T-cells that specifically recognize (i.e., bind) a peptide fragment with at least one epitope will continue to expand, thereby permitting ready identification.
  • T-cell eliciting epitopes of a myelin protein can be used as an alternative to using the entire protein. Additionally, one can prepare other fragments containing the epitope to enhance its ability to elicit a T-cell response.
  • a degenerate variant of the MBP DNA sequence is the same as that MBP sequence, except that the degenerate variant includes at least one nucleotide change which results in one or more alternative codons being used to encode the MBP amino acid sequence.
  • the degenerate variant includes at least one nucleotide change which results in one or more alternative codons being used to encode the MBP amino acid sequence.
  • a recombinant pox virus of the invention may include the MBP DNA sequence, except that the variant will include at least one nucleotide change which results in one or more alternative codons being used to encode the MBP amino acid sequence, whereby the alternative codon encodes a conservative amino acid, i.e. an amino acid which can substitute for another amino acid because of similar characteristics.
  • conservative amino acid substitutions include, e.g., valine for glycine, arginine for lysine, leucine for valine, serine for threonine, etc.
  • a conservative amino acid substitution by definition, will not substantially affect T-cell eliciting epitope(s) of MBP.
  • a recombinant pox virus of the invention may include the MBP DNA sequence, except that the variant will include at least one nucleotide change which results in one or more alternative codons being used to encode the MBP amino acid sequence, whereby the alternative codon encodes a non-conservative amino acid, i.e., an amino acid with different characteristics than the corresponding amino acid in the MBP amino acid sequence.
  • the non-conservative amino acid substitution does not substantially affect T-cell eliciting epitope(s) of MBP.
  • the recombinant pox virus may include deletions of the MBP DNA sequence, or insertion of foreign
  • a T-cell eliciting epitope of MBP in a polypeptide fragment is another type of MBP variant.
  • the recombinant pox viruses of the present invention can be administered to a suitable host using any acceptable route, including, for example, scarification and injection, e.g., intradermal, subcutaneous, intramuscular, intravenous or intraperitoneal.
  • a therapeutic composition of the present invention will suitably include one or more recombinant pox virus of the invention, which composition will typically be administered in unit dosage form to a suitable host in a sterile aqueous or non-aqueous solution, suspension or emulsion in association with a pharmaceutically-acceptable carrier such as physiological saline.
  • a pharmaceutically-acceptable carrier such as physiological saline.
  • Formulations for parenteral administration may also contain as common excipients polyalkylene glycois such as polyethylene glycol, oils of vegetable origin, hydrogenated napthalenes and the like. (See generally Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980).
  • the therapeutic compositions of the invention may be employed as the sole active agent in a pharmaceutical or can be used in combination with other compounds and/or therapies which serve to treat inflammatory demyelinating disease.
  • the amount of the recombinant pox virus in the therapeutic composition will vary depending on a number of factors, including the dosage of the virus administered, the virulence of the selected virus, and the route of administration.
  • a composition is provided in aqueous physiological buffer solution containing about 10 5 to 10 9 pfu for parenteral administration.
  • typical dose ranges will include about 10 5 to 10 9 pfu recombinant avipox or suipox virus, preferably fowl pox or swine pox.
  • the preferred dosage of the therapeutic composition is likely to depend on such variables as the type and extent of progression of the inflammatory demyelinating disease, the overall health status of the patient, the relative virulence and biological efficacy of the particular recombinant pox virus selected, the formulation of the composition excipients and its route of administration.
  • pox viruses have been developed as live viral vectors for the expression of heterologous proteins (Cepko et al., ( 1984); Morin et al., (1 987); Lowe et al., (1987); Panicali & Paoletti, (1982); Mackett et al., (1982)).
  • Representative fowlpox, swinepox virus and vaccinia are available through the ATCC under accession numbers VR-229, VR-363, and VR-325 respectively.
  • Genes that code for MBP or a T-cell eliciting epitope thereof are inserted into the genome of a pox virus such as e.g., fowl pox virus, swine pox or vaccinia in such a manner as to allow them to be expressed by that virus along with the expression of the normal complement of parent virus proteins.
  • a pox virus such as fowl pox virus, swine pox or vaccinia
  • the DNA donor plasmid contains the following elements:
  • a prokaryotic origin of replication so that the vector may be amplified in a prokaryotic host
  • a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector e.g., a gene encoding antibiotic resistance
  • donor plasmids for the introduction of multiple foreign genes into pox virus are described in W091 /19803, the techniques of which are incorporated herein by reference.
  • all DNA fragments for construction of the donor vector including fragments containing transcriptional promoters and fragments containing sequences homologous to the region of the parent virus genome into which foreign genes are to be inserted, can be obtained from genomic DNA or cloned DNA fragments.
  • the donor plasmids can be mono-, di-, or multivalent (i.e., can contain one or more inserted foreign gene sequences).
  • the donor plasmid preferably contains an additional gene which encodes a marker which will allow identification of recombinant viruses containing inserted foreign DNA.
  • marker genes can be used to permit the identification and isolation of recombinant viruses. These include genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos et al. (1 988); Falkner and Moss. (1988); Franke et al. (1 985), as well as genes such as the E. coli lacZ gene, that permit identification of recombinant viral plaques by colorimetric assay (Panicali et al.(1 986)) . Integration Of Heterologous DNA Into Pox Virus
  • Homologous recombination between donor plasmid DNA and viral DNA in an infected cell results in the formation of recombinant viruses that incorporate the desired elements.
  • Appropriate host cells for in vivo recombination are generally eukaryotic cells that can be infected by the virus and transfected by the plasmid vector. Examples of such cells suitable for use with a pox virus are chick embryo fibroblasts, HuTK143 (human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infection of cells with pox virus and transfection of these cells with plasmid vectors is accomplished by techniques standard in the art (Panicali and Paoletti, U.S. Patent No. 4,603, 1.1 2, WO89/03429, both references herein incorporated by reference).
  • recombinant viral progeny can be identified by one of several techniques. For example, if the DNA donor vector is designed to insert one or more foreign genes into the parent virus thymidine kinase (TK) gene, viruses containing inserted DNA will be TK " and can be selected on this basis (Mackett et al., ( 1982)). Alternatively, co-integration of a gene encoding a marker or indicator gene with the foreign genets) of interest, as described above, can be used to identify recombinant progeny.
  • TK thymidine kinase
  • One preferred indicator gene is the E. coli lacZ gene: recombinant viruses expressing ⁇ - galactosidase can be selected using a chromogenic substrate for the enzyme (Panicali et al.(1 986)).
  • a variety of methods can be used to assay the expression of the polypeptide encoded by the inserted gene. These methods include black plaque assay (an in situ enzyme immunoassay performed on viral plaques), Western blot analysis, radioimmunoprecipitation (RIPA), and enzyme immunoassay (EIA).
  • black plaque assay an in situ enzyme immunoassay performed on viral plaques
  • Western blot analysis Western blot analysis
  • EIA enzyme immunoassay
  • Antibodies that recognize a myelin protein such as, e.g., MBP, MBP-related proteins, or T-cell eliciting epitopes thereof, are commercially available or, alternatively, can be made by conventional techniques.
  • monoclonal antibodies against these MBP, MBP-related proteins, or T-cell eliciting epitopes thereof can be made by employing standard hybridoma technology (see, e.g., Kohler et al. ( 1 975); Kohler et al. (1976); Hammerling et al.
  • standard hybridoma technology see, e.g., Kohler et al. ( 1 975); Kohler et al. (1976); Hammerling et al.
  • anti-MBP antibody was quantified by ELISA. For example, microliter plates were coated with MBP ( 100 ng/well, SOURCE), ovalbumin ( 100 ng/well, Sigma), or 1 x10 7 PFU/well UV- inactivated V-Wyeth in PBS. The plates were blocked with 2% BSA in
  • a recombinant pox virus disclosed herein may, in some settings, elicit an undesirably strong immune response to the pox virus vector. Repeated administration of the same vector in subsequent boosts might result in undersirably rapid clearance of the virus from the host, not allowing sufficient time to express the myelin protein and boost the immune response to it. Thus while numerous "boosts" with recombinant pox virus of the invention are possible, repeated use of any one virus thereof may not always be preferred. The use of recombinant pox viruses from an antigenically distinct pox virus can in most cases minimize this problem.
  • the second and subsequent recombinant pox virus is selected from a different genus such as an orthopox, e.g. vaccinia, or a suipox.
  • pox virus which exhibits a suitable host range and/or tissue specificity.
  • pox virus can be selected whose primary host range is different than the animal that the gene delivery system is to be used in.
  • avipox such as, e.g., fowl pox
  • suipox such as, e.g, swine pox
  • the use of suipox would not be advantageous.
  • Attenuated strains of pox viruses may be desirable when another pox virus outside the host range of the host is not available.
  • highly attenuated strains of certain orthopox viruses such as vaccinia (MVA strain) may be used or alternatively, may be further modified by conventional genetic or chemical mutagenesis techniques to be even more attenuated or non-virulent in the normal host range.
  • the cell specificity of the pox virus of interest is one way to easily screen for infectivity and replication efficiency.
  • the gene encoding MBP was excised from a 2.2 kb cDNA fragment isolated from a human brain cDNA library [Kamholz et al., PNAS (USA) 83:4962-4966 (1986)].
  • pt3064 The resulting plasmid, designated pt3064, is used to make vT92, which contains the MBP gene under the control of the vaccinia virus 40K early/late promoter (Gritz et al., 1990) and the £. coli la cZ gene under the control of the fowlpox virus C1 promoter (Jenkins et al.,
  • This plasmid was deposited with the ATCC on March 22, 1 996 and given accession number 97491 .
  • the foreign sequences are flanked by DNA sequences from the Bam HI J region of the fowlpox virus genome (Jenkins et al., 1991 ).
  • a derivative of the POXVAC-TC (Schering Corp.) vaccine strain of fowl pox virus was used as the parental virus.
  • recombinant fowlpox virus was accomplished via homologous recombination between fowlpox sequences in the parental fowlpox virus genome and the corresponding sequences in pT3064 in fowlpox-infected chick embryo dermal (CED) cells transfected with pT3064.
  • Recombinant virus designated vT92
  • vT92 was identified and plaque-purified by growth on CED cells in the presence of Bluogal (Life Technologies; Gaithersburg, MD), a chromogenic substrate for / 9-galactosidase.
  • Insertion of the MBP gene into the fowlpox genome was confirmed by amplification of the inserted sequences by polymerase chain reaction (PCR). Expression of MBP was demonstrated by Western analysis using MBP-specific antisera.
  • the gene encoding for human MBP was derived from a 2.2 kb cDNA fragment isolated from a human cDNA library [21 ].
  • the resulting plasmid, designated pT1 1 5 containing the MBP gene under the control of the vaccinia virus early/late promoter [22] flanked by DNA sequences from the Hind III M region of the vaccinia genome.
  • flanking sequences include the vaccinia K1 L host range gene which is required for multiplication in human cells [23] .
  • the plasmid was deposited with the ATCC on March 22, 1996 and given accession number 97490.
  • a derivative of the New York City Health (NYCBH) strain of vaccinia was used as the parental virus in the construction of recombinant vaccinia virus.
  • This parental virus was designated vAbT33 (parent). This was deposited with the ATCC on May 1 5, 1989 and given ATCC No. VR-2240.
  • the parental virus lacks a functional K1 L gene and thus cannot efficiently replicate on rabbit kidney RK13 cells [24].
  • vaccinia virus was generated via previously described standard homologous recombination techniques. Briefly, homologous recombination was performed between vaccinia virus sequences in the vAbT33 (parent) genome and the corresponding sequences in RK13 cells infected with the parental virus and transfected with the pT1 15 vector.
  • the RK13 cell line is publicly available (ATCC: Accession No. CCL37) .
  • a recombinant vaccinia virus bearing the MBP gene was selected by growth on RK13 cells.
  • One recombinant vaccinia virus isolate included the entire MBP gene by Southern blot hybridization of restriction enzyme digests. The recombinant virus was designated vT1 5 (MBP). Animals
  • vT15(MBP) infected cell lysates by Western blot analysis using the polyclonal rabbit anti-guinea pig MBP R 120.
  • This polyclonal antibody binds human MBP.
  • the preparation of the antibody has been previously described ([25], A control recombinant vaccinia virus was prepared which includes the gene encoding the equine herpes virus gH was prepared using homologous recombination as described above. The resulting recombinant vaccinia virus was designated vAbT249 (control).
  • Vaccination with recombinant vaccinia viruses Eight marmosets were vaccinated with about 10 7 pfu of either vAbT249 (control) or vT15 (MBP) in 4 subcutaneous injections in the back. In some animals, the initial injection was followed eight weeks later by a booster injection of 10 9 pfu. In a single monkey, we observed generalized cowpox lesions and transient fever from 10- 14 days after first vaccination. There were no other detectable side- effects over this time interval. The pathological and immunological consequences of vaccinating the animals were evaluated by measuring pathological manifestations such as, e.g., the appearance and severity of skin lesions, anti-vaccinia T-cell activity, and antibody responses as described fully below.
  • pathological manifestations such as, e.g., the appearance and severity of skin lesions, anti-vaccinia T-cell activity, and antibody responses as described fully below.
  • PBMC Infected with recombinant vaccinia virus intracellularly express MBP
  • MBP MBP
  • VAbT249 control
  • MBP Expression was also monitored in vivo in PBMC every 2-3 weeks following vaccination.
  • Cytospin preparations (Shandon) were fixed in 50% ethanol, blocked with 1 % bovine serum albumin in phosphate buffered saline and stained with R 120 rabbit polyclonal anti guinea pig MBP (1 :200), after washing slides were incubated with peroxidase-conjugated anti rabbit IgG (Sigma). Color development was achieved using 3-amino-9-ethyl-carbazole (Sigma) and slides were counterstained with hematoxylin/eosin.
  • BLCL Monkey autologous B lymphoblastoid cell lines
  • vaccinia virus vT15 expressed the myelin protein in PBMC obtained from vaccinated animals. This was not seen with a control group of marmosets vaccinated with vAbT249 (control). In each animal, the period during which MBP was expressed varied between 15-45 days after the first or initial vaccination. Immune responses against MBP and vaccinia antigens T-cell responses were measured in a standard 72 hr. proliferation assay [13] using 3 [H]-thymidine incorporation in 10 5 freshly isolated PBMC plated in 96 well round bottom plates.
  • each plate contained in 200 ⁇ l/AIM V (Gibco-BRL) and one of the following: no addition (control); MBP 50 ⁇ g/ml; Proteolipid protein (PLP) 10 ⁇ g/ml; PHA 2.5 ⁇ g/ml; WM 0.1 % (wt/vol.); NYCBH vaccinia strain 2 ⁇ g/ml.
  • Stimulation indices were calculated as the ratio of unstimulated to stimulated PBMC.
  • the term "stimulation index" means the ratio of the amount of T-cell activity in PBMC that is detected in a biological sample exposed to a recombinant vaccinia virus of the invention to the amount of T-cell activity in PBMC not exposed to the virus. Animals were terminated by euthanasia; spleen and lymph nodes harvested and then tested in the T-cell proliferation assays.
  • MBP and vaccinia virus binding antibodies were followed every 14-21 days using a dot-blot filtration apparatus according to the manufacturer's instructions (Biorad) [13]. Briefly, 250 ng MBP or 500 ng NYCBH strain were adsorbed on 0.45 ⁇ m nitrocellulose filter membranes (Biorad), blocked and incubated in succession with a) serial dilutions of marmoset sera; b) anti-monkey IgG conjugated to peroxidase (Sigma, 1 :4,000 dilution); c) diaminobenzidine (Pierce) as substrate for color development.
  • Biorad dot-blot filtration apparatus according to the manufacturer's instructions [13]. Briefly, 250 ng MBP or 500 ng NYCBH strain were adsorbed on 0.45 ⁇ m nitrocellulose filter membranes (Biorad), blocked and incubated in succession with a) serial dilutions of marmoset sera; b) anti-monkey IgG conjugated to peroxidase (Sigma,
  • the vT15 (MBP) recombinant vaccinia virus alleviates EAE
  • Neuropathological examination was performed on one vaccinated control animal (animal no. 346-92) at the time of acute EAE (22 days after immunization and 5 days after onset of EAE, see Figure 1 A).
  • two animals vaccinated with vT1 5 (MBP) (animal nos. 344-92 and 353-92, see Figure 1 B) were studied 63 days after the first immunization.
  • pathology typical of acute EAE in C. jacchus [ 1 2, 1 31.
  • CNS white matter we observed multiple areas of perivascular infiltration comprised of mononuclear cells and macrophages. These manifestations were accompanied by prominent concentric demyelination and early gliosis.
  • vaccinia virus in some instances can have encephalitogenic properties (Paoletti et al. ( 1 993)). We therefore investigated whether vaccination with the
  • NYCBH strain of vaccinia virus could by itself augment this EAE in C. jacchus.
  • T-cell reactivity against MBP In two marmosets vaccinated with vT15 (MBP), transient and modest (i.e. stimulation index of 2) proliferative responses were observed prior to induction of EAE with WM (animal nos. 344-92 and 353-92, see Figures 2A and C) . This result indicates that vaccination with the recombinant vaccinia virus presents the autoantigen to the immune system in vivo. Following active immunization with WM, T-cell proliferative responses against MBP were observed by days 1 5 and 22 in two of the vAbT249 (control) vaccinated animals and by day 62 in the third control monkey.
  • T-cell proliferation against MBP did not develop in the vT1 5 (MAB) vaccinated monkeys up to 74 days after immunization.
  • MAB vT1 5
  • T-cell responses against MBP remained negative during the entire experiment. In no instance was T- cell reactivity against PLP detected in these experiments.
  • T-cell reactivity against vaccinia virus Strong T-celi proliferative responses against NYCBH were observed in both the experimental and control groups following either vaccination or immunization with WM (see Figures 2C and D) . This indicated that subcutaneous injection of the recombinant vaccinia viruses used herein efficiently stimulated cellular immune responses in C. jacchus. In animals later developing acute disseminated encephalomyelitis, anti- vaccinia T-cell responses increased.
  • T-cell reactivity against PLP No animal exhibited T-cell reactivity against PLP in response to immunization with WM. However, one animal developing EAE exhibited a strong proliferative response against PLP.
  • NYCBH strain of vaccinia virus were detectable 14-21 days following the first vaccination. This result indicates that, like T-cell responses, humoral immunity against vaccinia antigens had been induced by vaccination.
  • vaccinia virus vectors Presentation of antigens by vaccinia virus vectors is achieved in part via the endogenous pathway of antigen presentation in association with MHC class I antigens. This could stimulate the proliferation of regulatory T-cells, in particular CD8 + T-cells.
  • regulatory T-cells in particular CD8 + T-cells.
  • the alleviation of EAE observed in these experiments may be due to either immunological suppression of certain subsets of T-cells, or secretion of inhibitory cytokines by regulatory T- cells, or both.
  • These protective mechanisms may be negated by the encephalitogenic properties of vaccinia virus.
  • the recombinant fowl pox virus disclosed above can be used in place of the recombinant vaccinia virus, in order to ameliorate or delay the onset of an inflammatory demyelinating disease in a mammal, particularly domesticated animals, captive animals, or humans.
  • kits suitable for clinical or veterinary use Such a kit would include one or more recombinant pox virus of the invention in a pharmaceutically acceptable carrier, or cell or homogeneous population of cells which include the recombinant pox virus.
  • the cells or homogeneous population of cells can be part of a diagnostic kit whereby the cells or homogeneous population of cells intracellularly express a myelin protein, preferably MBP, MBP-related protein, or a T-cell eliciting epitope thereof, which cells or homogeneous population of cells are capable of binding T-cells derived from a host suspected of having or predicted to suffer from an inflammatory demyelinating disease as described herein.
  • a myelin protein preferably MBP, MBP-related protein, or a T-cell eliciting epitope thereof
  • T-cell eliciting epitope a myelin protein
  • the binding between the T- cells and the cells or homogeneous cells disclosed herein can be detected.
  • T cells responsive to myelin basic in patients with multiple sclerosis T cells responsive to myelin basic in patients with multiple sclerosis. Science, 247: 71 8-21 .
  • lymphocytes in multiple sclerosis determined by antigen-induced
  • Immunological self tolerance an analysis employing cytokines or cytokine receptors encoded by transgene or a recombinant vaccinia
  • viruses as vectors for studying T lymphocyte specificity and function.

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Abstract

L'invention concerne un poxvirus recombinant codant pour une protéine de la myéline, qui peut être utilisé pour provoquer des réponses immunitaires contre ladite protéine. La méthode comprend l'introduction d'une quantité suffisante de poxvirus pour présenter la protéine de myéline au système immunitaire, l'expression de ladite protéine et sa présentation au système immunitaire.
PCT/US1997/005217 1996-03-25 1997-03-25 Poxvirus recombinant codant pour la proteine de la myeline et destine a un usage therapeutique WO1997035997A2 (fr)

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AU26586/97A AU2658697A (en) 1996-03-25 1997-03-25 Recombinant pox virus encoding myelin protein for therapy

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1015034A1 (fr) * 1996-05-29 2000-07-05 University Of Southern California Construction et utilisation de genes codant des epitopes pathogeniques pour le traitement de maladies autoimmunes
US9944903B2 (en) 2006-10-16 2018-04-17 Genelux Corporation Modified vaccinia virus strains for use in diagnostic and therapeutic methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CLINICAL INVESTIGATION, vol. 96, no. 6, December 1995, pages 2966-2974, XP002038650 GENAIN, C.P. ET AL.: "Antiboby facilitation of Multiple Sclerosis-like lesions in a nonhuman Primate" *
NEUROLOGY, vol. 46, no. 2 suppl., February 1996, pages a220-a221, XP002038649 GENAIN, C. P. ET AL.: "Inhibition of allergic encephalomyelitis in marmosets by vaccination with recombinant vaccinia virus encoding for Myelin Base Protein" *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1015034A1 (fr) * 1996-05-29 2000-07-05 University Of Southern California Construction et utilisation de genes codant des epitopes pathogeniques pour le traitement de maladies autoimmunes
EP1015034A4 (fr) * 1996-05-29 2004-12-01 Univ Southern California Construction et utilisation de genes codant des epitopes pathogeniques pour le traitement de maladies autoimmunes
US9944903B2 (en) 2006-10-16 2018-04-17 Genelux Corporation Modified vaccinia virus strains for use in diagnostic and therapeutic methods
US10584317B2 (en) 2006-10-16 2020-03-10 Genelux Corporation Modified vaccinia virus strains for use in diagnostic and therapeutic methods

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