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WO2005092374A2 - Virus d'herpes simplex recombinant et leurs utilisations - Google Patents

Virus d'herpes simplex recombinant et leurs utilisations Download PDF

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Publication number
WO2005092374A2
WO2005092374A2 PCT/EP2005/003639 EP2005003639W WO2005092374A2 WO 2005092374 A2 WO2005092374 A2 WO 2005092374A2 EP 2005003639 W EP2005003639 W EP 2005003639W WO 2005092374 A2 WO2005092374 A2 WO 2005092374A2
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hsv
vaccine
antigen
cells
vaccine according
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WO2005092374A3 (fr
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Barbara Ensoli
Antonella Caputo
Peggy Marconi
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Istituto Superiore di Sanita ISS
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Istituto Superiore di Sanita ISS
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to the use of the herpes simplex virus (HSN) in a vaccine capable of inducing a CD8 + immune response in an animal and in a method of screening heterologous candidate antigens for use in vaccines.
  • HSN herpes simplex virus
  • Niruses are often used in vaccines, either in an attenuated form or in a fully functional form.
  • Viruses are often referred to as vectors and are frequently used to deliver antigens.
  • a large number of viral vectors are known for use in vaccines. These may include poxvirus- based vectors, alphavirus self- replicating vectors, adenovirus, and lentivirus, for instance. Many of these viral vectors are known for their capacity to induce in vivo strong Thl and CTL responses, as well as antibody titres against various antigens, such as HIV-1 gene products.
  • HSV vectors for use in prophylaxis against, for instance, viral infections show several advantages. HSV vectors have been shown to elicit strong and durable immune responses by various routes of inoculation; the viral D ⁇ A persists inside the host's cell nucleus as an episomal element, thus eliminating safety concerns arising from possible random integration of the viral genome into the host's D ⁇ A.
  • the present invention provides a vaccine capable of inducing a strong immune response when encoding a heterologous antigen.
  • CD8 immune response in an animal comprising replication-deficient HSV
  • HSV comprising polynucleotides encoding a heterologous antigen.
  • the polynucleotides are DNA, although RNA is also envisaged.
  • a heterologous antigen is an antigen that is not derived from HSV.
  • Cytotoxic T cell activation is part of the so-called cellular immune response, and leads to mature cytotoxic T lymphocytes (CTLs) specific for a particular antigen. This is part of the MHC1/CD8 + antigen processing, recognition and clearance pathway. Mature CTLs, aided by CD4 + Helper T cells, recognise antigens presented on an infected or cancerous cell, leading to the lysis of said cell.
  • CTLs cytotoxic T lymphocytes
  • the HSV having been introduced or administered to an animal as part of a vaccine, enters suitable cells, such as endothelial, mucosal or nerve cells, and proteins encoded within its genome are expressed.
  • the HSV genome encodes a heterologous antigen.
  • the expression of the antigen in the infected cell induces a CD8 immune response as the antigen is processed in the cell, such that a fragment or peptide thereof is presented on the cell surface in combination with a MHC Class 1 molecule.
  • immature CTLs with Class 1 -restricted antigen-specific T cell receptors and CD 8 molecules on their cell surfaces bind to the peptide-MHCl complex, leading to cytokine release, and CTL proliferation and differentiation.
  • the animal to which the vaccine is administered may be any animal capable of inducing such an immune response, but preferably a mammal, more preferably a mouse or a monkey, and most preferably a human.
  • the antigen when expressed in the host or patient induces an immune response, preferably a CD8 + response.
  • the antigen may be functional or non-functional protein or a fragment thereof, provided that it comprises at least one epitope and can, therefore, be bound by antibodies or raised to the antigen or lymphocytes specific for the antigen.
  • the vaccine may be administered by any means known in the art.
  • the vaccine is adapted for the means of administration.
  • the vaccine may be suitably formulated together with a pharmaceutically acceptable carrier or diluent, such that it may be administered orally, transdermally, or through a mucous membrane such as the nose or rectum. . It is, however, preferred that the vaccine is administered intravenously, intraperitoneally, subcutaneously, intramuscularly or intradermally. It will be understood that the formulation of the vaccine is dependent upon the preferred means of administration.
  • the vaccine may be administered as a so-called "single-shot" vaccine, wherein the vaccine is preferably administered once to the same animal.
  • the vaccine can also be administered repeatedly as part of a repeated vaccination regimen.
  • Such a regimen may be a homologous or a heterologous prime-boost regimen.
  • the vaccine may be administered at separate times, the first occasion being the "prime” and subsequent occasions being the "boosts".
  • the vaccine may be administered together with, but preferably separate from, a further means of inducing an immune response.
  • This further means of inducing an immune response preferably comprises the same antigen as the present vaccine, which may be in the form of a protein or peptide.
  • this further means of inducing an immune response may comprise a polynucleotide encoding the antigen, for instance a plasmid or a non-HSV viral vector, such as an adenovirus, poxvirus, lentivirus or retro virus.
  • the HSV due to the size of its genome, can accommodate more than one inserted gene, in general. Accordingly, the HSV of the present invention may encode more than one antigen, although this depends on the size of the genes.
  • the further antigens may be derived from the same source as the first antigen, for instance multiple epitopes from the same protein or, alternatively, different proteins derived from the same source. For instance, should the vaccine be directed towards HIV, then the antigen may be the entire ENV HIV protein or any of its peptides, and the further antigens may be either separate peptides from the same ENV (clades A, B or C) protein or may be derived from other HIV proteins such as the GAG, REV, POL or NEF proteins.
  • the antigens encoded by the HSV may also be derived from separate sources.
  • one of the antigens may be derived from HTV, whilst the other may be derived from another (non-HSV) virus, an intracellular bacterium or may be a tumour-associated antigen (TAA).
  • TAA tumour-associated antigen
  • the HSV may encode one or more antigens as well as any number of further proteins, such as Cytokines, that serves to enhance or regulate the immune response to the antigen or antigens.
  • the HSV may encode a co-factor or inhibitor that may have a regulatory effect on the patient's immune response or other biological processes, such as cell cycle.
  • the HSV may also encode an enzyme that has an activity in the infected cell once transcribed, such as a suicide gene.
  • the HSV according to this aspect of the present invention is replication-deficient or defective. Accordingly, the HSV is not capable of copying or replicating itself in its entirety once it has entered the host cell. Therefore, once the virus has entered the host cell, its lifecycle is effectively over and further host cells cannot be infected as no "daughter" virions are produced. Accordingly, it is preferred that the HSV is substantially or essentially avirulent in the patient. In particular, it is preferred that the HSV is unable to trans-induce immediate-early gene expression.
  • the HSV may be rendered replication-incapable by a number of means known in the art.
  • the impairment of replication is preferably by mutation or alteration of the genes or promoters responsible for HSV replication. Such a mutation may be by insertion, deletion or rearrangement, for instance by U. V. knock out.
  • the vector is rendered replication-deficient by replacement of the genes or promoters responsible for HSV replication by another gene.
  • the virulence, and preferably the toxicity, of the HSV virus is substantially reduced, and preferably completely eradicated, by the step of rendering the HSV replication-deficient, it will be appreciated that targeted mutation or replacement of the viral genome, or parts thereof, is preferred so that the remainder of the HSV genome is retained, thus helping to induce a stronger immune response against the HSV, which preferably aids an immune response to the HS V-encoded antigen via a synergistic mechanism.
  • one or all of the following immediate-early genes, ICP4, ICP22 and ICP27 are mutated in such a way that their expression is prevented or that the protein expressed is non-functional.
  • the VHS (Wirion Host Shutoff) structural protein may also be mutated with the same effect.
  • a preferred embodiment is the triple mutant ICP4 " , ICP22 “ and ICP27 " , which may also, optionally, include a mutated VHS gene (VHS " ).
  • the heterologous polynucleotides such as those encoding the antigen or further genes (which may include a further antigen or a cytokine or suicide gene), for instance, are provided as independent expression cassettes, inserted at different loci in the HSV genome, and preferably at HSV genes that are responsible for immediate early protein expression, preferably also rendering the HSV replication-deficient.
  • the HIN-1 env gene may be inserted in the US1 locus (encoding ICP22 protein) and the HIN-1 tat gene may be inserted in the UL41 locus, and so on.
  • the backbone is represented by the ICP4-ICP22-ICP27- mutant HSV-1.
  • the HSN comprises polynucleotides encoding more than one heterologous gene (for instance an antigen and a cytokine)
  • these heterologous genes are under the control of separate promoters in separate independent expression cassettes.
  • each independent expression cassette is used to mutate, disrupt and preferably replace an immediate early gene, and preferably UL13, but most preferably one of ICP4, ICP22, ICP27 and UL41 (encoding the NHS protein of HSN).
  • Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47 (see Krisky DM et al., Gene Ther. 1998 Dec;5(12):1593-603.).
  • the UL13 locus of HSV-1 is in the position 28502-26946 (complementary strand, see the ⁇ C_001806 NCBI reference and SEQ ID NO. 1).
  • the UL 13 is given separately as SEQ TD NOS. 2 and 3.
  • the UL41 locus (encoding VHS protein) is found at 92637-91168 of the above sequence, and also as SEQ ID NO. 4.
  • the UL54 locus (encoding ICP27 protein) is found at 113734-115272 of the above sequence, and also as SEQ ID NO. 5.
  • the first RSI locus (encoding ICP4 protein) is found at 131128-127232 of the above sequence, and also as SEQ TD NO. 6.
  • the second RSI locus (there are two of them, localised in the two short repeated regions of the HSV-1 genome, both encoding the ICP4 protein) is found at
  • ICP22 protein is found at 132644-133906 of the above sequence, and also as SEQ ID NO. 8.
  • the US4 locus (encoding gG protein) is found at 136744-137460 of the above sequence, and also as SEQ ID NO. 9.
  • the US5 locus (encoding gJ protein) is found at 137731-138009 of the above sequence, and also as SEQ TD NO. 10.
  • the HSV is a mutant, wherein at least one of the following loci are mutated: UL13, UL41 (encoding VHS protein), UL54 locus (encoding ICP27 protein), US1 (encoding ICP22 protein), US4 (encoding gG protein), US5 (encoding gJ protein) and either or both of the two RSI loci (encoding ICP4 protein).
  • these loci are mutated, disrupted or replaced, preferably by insertion of heterologous nucleotides encoding proteins that it is desired to express, as discussed elsewhere.
  • the vaccine is preferably capable of inducing a CD8 immune response in an animal, the vaccine comprising replication-deficient HSV, the HSV comprising polynucleotides encoding a heterologous antigen, where-in the HSV is mutated at at least one of the following loci: UL13, UL41 (encoding VHS protein), UL54 locus (encoding ICP27 protein), US1 (encoding ICP22 protein), US4 (encoding gG protein), US5 (encoding gJ protein) and either or both of the two RSI loci (encoding ICP4 protein).
  • loci UL13, UL41 (encoding VHS protein), UL54 locus (encoding ICP27 protein), US1 (encoding ICP22 protein), US4 (encoding gG protein), US5 (encoding gJ protein) and either or both of the two RSI loci (encoding ICP4 protein).
  • the HSV is preferably UL13-, UL41-, UL54-,US1-,US4-,US5-RS1-, and preferably a combination of at least two of these.
  • the HSV comprises mutations in any of SEQ ID NOS. 3, 4, 5, 6, 7, 8, 9 or 10.
  • mutant HSV which is UL54-, US1- and RS1-, where the HSV comprises mutations in SEQ ID NOS 5, 6, 7 and 8, i.e. 5, 8 and 6 or 7.
  • the HSV is preferably rendered replication-defective by incorporating non-reverting mutations into mandatory viral genes, such that the HSV maintains the immunogenicity of wild-type HSV, but is much safer.
  • Non-reverting mutations are preferred in comparison with mutations that are capable of reversion. Mutations that are capable of reversion usually involve only minimal changes in the original DNA sequence, such as transitions, transversions, or frame shifts. Thus, whilst any mutation or disruption of the viral genome that is sufficient to render the HSV substantially avirulent is preferred, it is more preferable that the mutation is large enough to render the HSV substantially avirulent permanently.
  • HSV mutants showed a reduced immunogenicity, due to their inability to replicate and to spread in the host, but still retain the capability to infect a wide range of tissues and host species.
  • these recombinant replication-deficient viral vectors can sustain high expression of any foreign or heterologous genes, such as an antigen to which the vaccine is directed, under homologous or heterologous promoters, such as HSV-1 or HCMV, respectively (references 47 and 48 in Example 5).
  • homologous or heterologous promoters such as HSV-1 or HCMV, respectively.
  • their large genome can accommodate a relatively large antigen, or even multiple antigens (reference 49 in Example 5), which can be simultaneously expressed.
  • Recent studies also indicate that pre-existing immunity against HSV infection does not compromise its efficacy as a vaccine vector (references 50 and 51 in Example 5).
  • the DNA or polynucleotides encoding the antigen is preferably under the control of a promoter and even more preferably under the additional control of an enhancer.
  • Such genetic elements are well known in the art.
  • the DNA encoding the antigen may form together with such elements an expression cassette.
  • the DNA or polynucleotides are under the control of an HSV immediate early promoter.
  • the antigen is under the control of a promoter such as the human Cytomegalovirus (CMV) promoter.
  • CMV Cytomegalovirus
  • the promoter is inducible, preferably by an extrinsic factor. This allows the administrator of the HSV, such as a health official, to induce expression of the antigen or heterologous DNA encoded by the HSV when required, which may be some time after infection of the patient by HSV.
  • the first promoter for instance, may lead to high levels of expression of the first gene on infection of the patient by HSV, i.e. on administration of a vaccine comprising the HSV of the present invention, whilst a second promoter controlling expression of the other gene, is induced at a later stage, for instance as part of a later "boost” as further discussed below.
  • the inducer could be taken orally, for instance, which may be more acceptable than the preferred means of administration of the HSV itself, which may be intravenously.
  • the heterologous DNA encoded by the HSV is under the control of at least one, and preferably at least two, inducible promoters.
  • the inducer of the promoter is an extrinsic factor.
  • a suitable example of such a promoter and inducer system is the Tet-on/Tet- off promoter system, which is induced by the absence of tetracycline in the diet.
  • expression of the antigen or heterologous DNA may be delayed under the control of a suitable promoter such as the Tet-on/Tet-off promoter, this is not generally preferred.
  • the promoter used results in an immediate and high level of antigen protein or peptide expression, such that the antigen is preferably co-temporaneously expressed with an immune response raised against the HSV vector itself.
  • the antigen or heterologous DNA is expressed as soon as possible after the vaccine is administered or introduced into the animal.
  • HSV which itself raises an HSV-directed immune response
  • antigen-specific immune response It is thought that the presence of HSV viral vectors may result in a stronger antigen-specific immune response, as both are likely to raise MHC1/CD8 cellular immune responses. Therefore, it is preferred that both the anti- HSV and the antigen-specific response are raised at the same time.
  • the HSV comprises polynucleotides encoding more than one heterologous gene (for instance an antigen and a cytokine)
  • these heterologous genes are under the control of separate promoters in separate independent expression cassettes.
  • each independent expression cassette is used to mutate, disrupt and preferably replace an immediate early gene, and preferably UL13, but most preferably one of ICP4, ICP22, ICP27 and UL41 (encoding the VHS protein of HSV).
  • antigens it will be understood that the present invention relates to any heterologous DNA or genetic material that it is wished to be expressed. Thus, it will be understood that the terms are interchangeable, unless otherwise apparent.
  • the antigen may be derived from a number of different sources, as mentioned above.
  • the antigen may be derived from a disease-causing agent.
  • This disease-causing agent may be a fungus or a parasite, such as Plasmodium ssp.
  • the antigen may be derived from a virus, an intracellular bacterium, or a tumour.
  • Suitable viruses include HIV, in which case the antigen is preferably derived from the POL, ENV (clades A, B or C), REV or NEF HIV proteins or peptide fragments thereof. It is particularly preferred that the antigen is derived from the HIV GAG protein.
  • Example 6 shows that whilst recombinant HSV-1 derived vaccines may only be weak inducers of CD4 T helper dependent antibody responses, they are capable of activating or inducing efficient long-term CD8 T cell responses.
  • T0H:gag and TO-tat replication-defective HSV-1 recombinant vectors were able to elicit a Gag- and Tatspecific immune response respectively in immunized mice, although the breadth of the response was different depending on the site of inoculation.
  • the antigen is preferably not derived from SIV, unless the HSN further encodes a cytokine or suicide gene, or both, as discussed elsewhere.
  • the antigen is derived from an intracellular bacterium. These may be, for instance, Listeria, Salmonella or M. tuberculosis bacteria.
  • the antigen may be derived from a tumour or pre-cancerous cell which may be in the process of becoming cancerous.
  • the antigen is a tumour-associated antigen (TAA), which are well known in the art.
  • the tumour may be a neoplasia, glioma or glioblastoma and is preferably derived from cancerous endothelial or C ⁇ S cells.
  • the antigen is derived from any cancerous cell, including those associated with AIDS-related dysfunctions, such as Kaposi's sarcoma, as discussed below.
  • the HSN is He ⁇ es Simplex Virus - type 2 and, even more preferably, Herpes Simplex Virus - type 1, also known as HHV (Human Herpes Virus).
  • the HSV further encodes a cytokine.
  • Suitable cytokines will be known to the skilled person. However, these may, preferably, include Interleukins or Colony- Stimulating Factors, in particular those that promote a Thl Type response in Macrophages, ⁇ K Cells, and/or induction of IF ⁇ -Gamma production.
  • cytokines IL-12 and GM-CSF are particularly preferred.
  • Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent stimulator of macrophages and Dendritic Cells which are important antigen-presenting cells involved in the induction of immune response.
  • GM-CSF Granulocyte macrophage-colony stimulating factor
  • IL-12 is a heterodimeric Thl cytokine with strongly immunomodulatory properties that promotes the proliferation of T cells, ⁇ K cells and tumour-infiltrating lymphocytes (TIL cells).
  • TIL-12 can induce a cascade of other cytokines and chemokines which possesses significant antiangiogenic properties.
  • the HSV comprises D ⁇ A encoding a cytokine as well as an antigen. It is particularly preferred that when the antigen is derived from SIN, the HSN also comprises D ⁇ A that encodes a cytokine.
  • cytokine is expressed locally in the relevant cell, therefore reducing the need for systemic administration.
  • the HSV comprises D ⁇ A encoding a suicide gene.
  • the HSV comprises D ⁇ A encoding a suicide gene, an antigen and a . cytokine.
  • the suicide gene may be a gene encoding viral or bacterial enzymes that converts an inactive form of a drug ("prodrug”) into toxic anti-metabolites capable of inhibiting nucleic acid synthesis, for example.
  • Suicide genes code for enzymes that convert nontoxic compounds (prodrugs) into toxic products.
  • Gene therapy with suicide genes is considered one of the most powerful approaches for cancer treatment: tumor cells transduced with suicide genes can be eliminated upon treatment with the prodrug.
  • Many suicide gene therapy approaches have been successfully used in animal models of cancer, and are currently being tested in clinical trials.
  • the most preferred are the HSVltk and the cytosine deaminase (CD) genes, as these are thought to be the most potent and widely used.
  • the suicide gene is a humanized suicide gene, as these are thought to have an improved cytotoxic activity.
  • Virus-originated HSV-TK gene is different from that of mammals. Its product, thymidine kinase, is able to metabolize the nontoxic prodrug, GCV, into a monophosphate derivative, then phosphorylate it further into GCV triphosphate. This metabolite is incorporated into replicating DNA strands and acts as both a DNA synthesis inhibitor and a cell cycle blocker, finally leading to cell apoptosis and cell death.
  • the therapeutic effect of this system is also based on a "bystander effect” whereby HSV-TK gene modified cells are toxic to nearby unmodified cells when exposed to the antiviral drug GCV.
  • the mechanism underlying this "bystander effect” is complex, but the primary mechanism is thought to be "metabolic cooperation” involving formation of gap junctions between cells that permit the passage of small molecules from one cell to another (Reference 39 from expt 2).
  • the suicide gene is the HSV-1 tk gene, as not only is “native" to the HSV, thus obviating any potential complications with the HSV genetic machinery, but the suicide gene is already present in HSV-1, so it does not need to be inserted by a user, thus simplifying the recombination process.
  • the skilled person will appreciate that some care must be taken to ensure that the HSV-1 tk gene is not mutated or disrupted, so that the functional enzyme is produced.
  • the animal is administered Ganciclovir at a suitable point, preferably co- temporaneously with the administration of the vaccine.
  • Ganciclovir is relatively non-toxic. It will be appreciated that suitable amounts of Ganciclovir will need to be administered, such that sufficient Ganciclovir is available for conversion into the toxic form in all HSV infected cells.
  • a further example of a suitable suicide gene is the bacterial cytosine deaminase (CD) system, for instance the Escherichia coli cytosine deaminase/5-fluorocytosine (CD/5-FC) system.
  • CD bacterial cytosine deaminase
  • CD/5-FC Escherichia coli cytosine deaminase/5-fluorocytosine
  • the TK system confers sensitivity to the respective pro-drug and the cytotoxic effect acts synergistically with combined expression of cytokines (references 16 and 40 in Experiment 2). Accordingly, it is preferred that the suicide gene and the cytokine gene are expressed co-temporaneously and, furthermore, that they are preferably expressed at the same time as the antigen. Thus, it is also preferred that at least two and, preferably three, of these genes are within different independent expression cassettes.
  • the vaccine further comprises DNA encoding an angiogenic inhibitor.
  • a vaccine capable of inducing an anti-cancer effect in an animal, comprising replication-deficient HSV whose DNA encodes an angiogenic inhibitor, a cytokine and a suicide gene.
  • the effect comprises an anticancer CD8 + immune response.
  • the HSV may also encode an antigen, preferably a tumour-associated antigen.
  • the vaccine preferably also comprises DNA encoding connexin 43 (CX43) which is involved in the formation of gap junctions, as this can increase the efficacy of the suicide gene, in particular the TK suicide gene, by enhancing bystander killing (references 2 and 29 from Experiment 2).
  • CX43 connexin 43
  • the antitumour response is selective and attacks the primary tumour, inhibits metastasis, prevents recurrence, and does not promote drug resistance.
  • Preferred anti-angiogenesis factors include angiostatin, kringle 5 (K5), or endostatin.
  • Angiogenesis is a complex process that includes endothelial cell proliferation, migration and differentiation, degradation of extra-cellular matrix, tube formation, and sprouting of new capillary branches. This process is tightly regulated by angiogenic factors and an unbalance between angiogenic stimulators and inhibitors leads to progression of many diseases, including tumour growth (Reference 14 from expt 2)
  • Angiostatin is one of the first identified endogenous specific inhibitors of tumour- related endothelial cell proliferation; it contains the first four disulfide-linked structures of plasminogen (Pgn), known as kringle structures (Reference 8 from expt 2). Smaller fragments of angiostatin display differential effects on suppression of endothelial cell growth but integrity of the kringle structures of angiostatin is required to maintain its inhibitor potency (References 25, 31 from expt 2).
  • the anti-anigiogenesis factor is Angiostatin or a functional variant thereof, wherein the integrity of the kringle structures of Angiostatin is retained.
  • Kringle 5 is another fragment of Pgn that, although related to the other four kringles in Pgn, is not present in angiostatin.
  • K5 is a specific inhibitor of endothelial-cell proliferation and appears to be more potent than angiostatin (Reference 7 from expt 2).
  • the combination of angiostatin and K5 exerts a synergistic inhibitory effect on endothelial cell proliferation.
  • endostatin a proteolytic fragment of type XVIII was identified: endostatin (Reference 32 from expt 2).
  • the identification of agents that inhibit angiogenesis represents a potential therapeutic approach for the treatment of solid tumours.
  • an angiogenic switch has been hypothesized to be responsible for transition from a slow, dormant phenotype to a faster, more aggressive one, although recent studies have demonstrated the ability of even a few tumour cells to recruit neovessels.
  • Endothelial cell proliferation, migration and tube formation are critical early events during angiogenesis, and their inhibition would be expected to affect the angiogenic process.
  • angiostatic proteins expressed by HSV vectors inhibit endothelial cell proliferation, migration and tube formation in vitro and reduce tumour angiogenesis in vivo.
  • Endostatin is another protein with a potent angiostatic activity (Reference 12 from expt 2).
  • Anti-tumour immunotherapy approaches are also rapidly evolving as we increase our understanding of the molecular events involved in the host's antitumor response (Reference 12
  • One of the causes of the tumour establishment may also be regarded as the outcome of the absence or ineffectiveness of an anti-tumour immune response.
  • a sort of "blindness” for neoplastic cells has been shown that in some cases “sight” can be restored by helping the immune system to recognize a tumour and to mount an effective reaction against it (Reference 33 from expt 2).
  • One way of doing this is offered by the use of cytokines released by the transduced tumour cells (Reference 9, 20 from expt 2).
  • hiterleukin 12 (IL12) is preferred.
  • suicide genes approach increases the possibility of rendering cancer cells more sensitive to chemotherapeutics or toxin agents (Reference 21, 36 from expt 2).
  • Most suicide genes currently under investigation mediate sensitivity by encoding viral or bacterial enzymes that convert inactive form of a drug ("prodrug”) into toxic anti-metabolites capable of inhibiting nucleic acid synthesis, as discussed above.
  • suicide genes are the viral thymidine kinase (TK) and the bacterial cytosine deaminase (CD) discussed above.
  • GM-CSF Granulocyte macrophage-colony stimulating factor
  • TK suicide gene increases the anti-tumour effects leading to prolonged survival and partial protection against a subsequent tumour challenge; this effect may be due to the combination of HSVtk/GCV- induced tumour cell death and tumour GM-CSF production attracting a greater number of host APCs that take up antigens derived from the dying tumour cells and cross-present them to the host's immune system (Reference 5 from expt 2).
  • a vaccine comprising HSV comprising DNA encoding both HSV tk and GM-CSF, is particularly preferred.
  • the activities of the various genetic elements encoded by the HSV are synergistic.
  • An example of this is the HSV tk enzyme in combination with GM-CSF, the effects of which are greater than the sum of their separate parts.
  • a further preferred example is that, in the case that the vaccine is directed to a tumour and the HSV encodes an angiogenic inhibitor, a cytokine and a suicide gene, it is preferred that the anti-tumour effect of the vaccine is greater than three separate vaccines comprising only one of these elements. It is preferred that the synergy applies in all aspects and embodiments of the invention according to the present application, where appropriate. In particular, it is preferred that the combination of expression of an antigen with a cytokine and/or with a suicide gene, is synergistic.
  • genetic element it is meant a gene, encoding a protein having a functional effect in the patient.
  • the genetic element may be heterologous to the HSV, such as an antigen, but it may also be homologous to HSV, i.e. derived therefrom, such as the tk gene.
  • the present invention also provides a method for screening putative vaccines comprising a candidate antigen in a non-human animal, the method comprising administering said vaccine to said animal and determining whether an immune response is successfully elicited to the antigen by subsequently administering a pathogenic amount of HSV comprising a polynucleotide encoding the antigen.
  • the antigen is first administered by means other than by replication- defective HSV, preferably by means of a plasmid comprising DNA encoding the antigen.
  • the putative vaccine is not replication-defective HSV, but may, preferably, be a plasmid encoding the antigen.
  • the HSV used is replication competent, so as to provide a suitably strong challenge to the host animal. It will be appreciated that if the challenge to the host is not strong enough to kill a non-immunised host, then the model will not be as efficient at screening for antigens, as further tests on the host will be required to determine whether any initial protective immunity was raised against the antigen.
  • the animal is a mouse.
  • the HSV may, preferably, be any HSV, including HSV-1 or HSV-2.
  • the antigens are as described above, although HTV and SIV viral antigens are particularly preferred.
  • HSV-2 it is particularly preferred that the VHS locus is mutated, preferably by deletion, although this should, preferably, not lead to a significant reduction in the replicative ability of the HSV.
  • a host is first vaccinated with a candidate antigen, where the antigen is a potential candidate for use in a vaccine regimen. To test whether the host has raised an immune response, that preferably leads to long-lasting protective immunity, the host is then further challenged with a replication competent HSV virus, which has been manipulated to express the antigen.
  • the antigen is a heterologous, non-HSV, antigen.
  • the host is preferably highly susceptible to HSV infection and dies when injected with a lethal dose of the replication-competent virus. However, if the host has previously raised an immune response to the antigen, then HSV infected cells, which express the antigen, will be recognised by the immune system and destroyed, by CTLs, for instance. However, if no immune response was previously raised, then the host will die, indicating that the candidate is not particularly suitable for use in vaccines, at least in that host. Suitable antigens can then be used in vaccine trials in other hosts, such as humans.
  • a host is first vaccinated with a candidate antigen, where the antigen is a potential candidate for use in a vaccine regimen.
  • the host is then further challenged with a replication competent HSV virus, which has been manipulated to express the antigen.
  • the antigen is a heterologous, non-HSV, antigen.
  • a host for instance a mouse, is first vaccinated against the candidate antigen.
  • This may be by any means known in the art, except that replication-deficient HSV are, preferably, not to be used.
  • the antigen may be delivered in the form of a plasmid comprising DNA, encoding the antigen, or by a viral vector, such as an adenovirus or pox virus.
  • the antigen may be delivered in its native state. If the antigen is a protein, it may be injected, for instance, directly into the mouse as a protein, rather than as DNA encoding the antigen.
  • the HIV Tat protein is used.
  • the mouse is then further challenged with a lethal dose of replication competent HSV encoding Tat, for instance by means of an HTV expression cassette inserted into the genome of the HSV virus under the control of a suitable promoter, as discussed elsewhere in the application.
  • a lethal dose of replication competent HSV encoding Tat for instance by means of an HTV expression cassette inserted into the genome of the HSV virus under the control of a suitable promoter, as discussed elsewhere in the application.
  • the mouse's immune system should respond to the HSV- Tat infection. This response will be detectable from the sera of the mouse, for instance by the increased presence of lymphocytes. If particularly successful, the mouse will rapidly clear the introduced HSV and antigen.
  • the mouse should die in 10-15 days.
  • HSV-1 or HSV-2 expressing the antigen may be used.
  • the antigen is preferably an HTV antigen, as discussed above, but particularly preferred are tat, gag (clade B), and env (clades A, B, and C).
  • the antigen is introduced according to a previously described method (Marconi et al., Proc. Natl. Acad. Sci. USA, 1996) in a HSV locus which is not essential for HSV-1 and HSV-2 replication (e.g. UL41, US4, US5).
  • HSV locus which is not essential for HSV-1 and HSV-2 replication (e.g. UL41, US4, US5).
  • These recombinant viruses are able to replicate and to induce disease and death in mice after inoculation of a lethal dose by systemic and mucosal routes. Survival of immunized mice 10-15 days after challenge with a lethal dose of recombinant HSV-1 expressing the vaccine antigens is the efficacy endpoint.
  • HSV-based challenge system is based on HSV-2, or genital herpes, which is more pathogenic in the genital tract as compared to HSV-1.
  • the present invention also provides HSV comprising a mutation in the UL13 protein kinase encoded by HSV, the mutation reducing levels of the HSV ICPO protein.
  • the HSV comprises a deletion in the UL13 locus of HSV-1 that encodes for protein kinase.
  • the UL13 sequence is in the viral genome in the locus that goes from position 26946 to 28502 of PubMed nucleotide sequence NC_001806 rwww.ncbi.nlm.nih. eov).
  • SEQ ID NO. 3 and the deletion is in the coding sequence where are deleted 164 bp from Sphl- 27508 to EcoRV-27672.
  • SEQ ID NO. 2 The nucleotide sequence of UL13 is given in SEQ ID NO. 2 and protein sequence is given in SEQ ID NO. 3.
  • SEQ ID NO. 3 is the nucleotide sequence for the HSV-1, taken from PubMed nucleotide sequence NC_001806.
  • the deletion is accomplished by insertion of non-native expression cassette encoding the Escherichia coli lacZ gene.
  • the expression cassette containing LacZ can be substituted by another non-native expression cassette encoding other gene of interest.
  • the new deletion in the mutated vectors increases the capacity of the vector to contain another non-native expression cassette and reduces the residual toxicity of the multiple mutated HSV vector.
  • restricted infected cells accumulate reduced levels of regulatory protein ICPO and several late viral proteins.
  • UL13 is involved in the posttranslational modification of the immediate early gene ICPO.
  • ICPO is phosphorylated by the UL13 protein kinase.
  • a preferred plasmid comprising the UL13 mutant is SK131acZ, as described in Example 10.
  • the present invention provides a vector comprising a deletion in the UL13 sequence of HSV-1 encoding a protein kinase, between position 27508 and 27672 of SEQ ID NO. 1, to provide a mutant lacking UL13 or a function variant thereof, thereby reducing levels of regulatory protein ICPO and several late viral proteins. Therefore, the mutant preferably cannot encode the protein according to SEQ ID NO.3.
  • a functional variant is a protein having substantially the same sequence as the reference sequence, except that it may comprise a number of mutations or amino acid changes, whilst still retaining substantially the same functional activity, preferably 50% or more activity.
  • the mutation may affect the coding sequence of UL13, so that the protein encoded lack activity due to imprecise folding, for instance, or the mutation may effect the promotion of expression of the UL13 protein, whether fully functional or otherwise.
  • the mutant has either none or reduced UL13 activity, whether this is due to reduced expression, reduced functionality of the expressed protein, or both.
  • an HSV vaccine according to the present application may be used a "single-shot" vaccine, making it more acceptable to the vaccinee, cheaper to produce and results in a vaccination regimen that is easier to administer.
  • the present invention further provides HSV transformed according to the present invention or for use in a vaccine or screening method of the present invention.
  • the present invention also provides a host non-human animal or transformant to which the present anti-pathogen or anti-cancer vaccine invention has been applied.
  • This may be an animal to which a vaccine according to the present invention has been administered, or animal which has been used in a screening method according to the present invention.
  • the animal is a mouse or rat.
  • HSV is a complex human neurotrophic virus with several characteristics which make it attractive as a vector for gene therapy (reference 6 from Example 1). Firstly, HSV replication-deficient mutants have been generated by the inactivation of genes essential for the virus life cycle. Furthermore, HSV replication deficient viruses efficiently infect almost all cell types at a relatively low multiplicity of infection.
  • HSV has a large genome (152 kb) which can accommodate up to 50 kb of exogenous DNA, thus allowing incorporation of multiple transgenes.
  • High titer stocks of replication deficient vectors can also be generated using cell lines which complement the essential HSV functions in trans. Indeed, highly deficient vectors due to the deletion of multiple essential genes have been created. These vectors show minimal cytotoxicity when infecting cells in vitro and in vivo (reference 24, 28 from Example 1).
  • Kaposi's sarcoma and cervical cancer
  • lymphomas cancers of the immune system known as lymphomas.
  • These cancers are usually more aggressive and difficult to treat in people with AIDS.
  • Signs of Kaposi's sarcoma in light-slrinned people are round brown, reddish, or purple spots that develop in the skin or in the mouth. In dark-skinned people, the spots are more pigmented.
  • FGF is one of the prominent cytokines expressed by AIDS-KS cells.
  • the ability of FGF to induce these lesions is augmented (in a synergistic fashion) by the HIV protein Tat, which is secreted by HIV-infected cells ( Reference 27 from expt 3). During acute HIV infections, Tat is released from infected cells.
  • Tat stimulates HIV gene expression, the growth of cells derived from Kaposi's sarcomas, angiogenesis and the promotion of tumour metastasis, the development of lymphoid hyperplasia, the secretion of TGF, TNF alpha and beta, IL-6, the malignant transformation of keratinocytes, the inhibition of IL-2 and IL-2 receptor gene expression, and the inhibition of the anti-viral alpha/beta interferon system.
  • Tat is known to activate endothelial cells and to be a powerful angiogenic growth factor.
  • Expression of Tat in transgenic mice induces Kaposi sarcoma-like lesions, squamous cell papillomas and carcinomas, adenocarcinomas of skin adnexa glands, and B-cell lymphomas.
  • Very small amounts of extracellular Tat may mimic VEGF by activating its receptor. This would explain a number of AIDS-related dysfunctions associated with endothelial cells, such as Kaposi's sarcoma, arteriopathy, and intravascular coagulopathy or hypercoaguloability of the blood.
  • the present invention also provides, in a further aspect, a therapy for inherited disorders with neurological implication, such as Tay-Sachs (TS) disease, which requires an active enzyme to be produced in the central nervous system to restore the cellular dysfunction caused by the defected gene.
  • TS Tay-Sachs
  • Hex hexosaminidase
  • HSV-T0 ⁇ Hex Herpes simplex vector encoding for the hexosaminidase (Hex) A alfa-subunit (HSV-T0 ⁇ Hex) and delivered it into the internal capsule of the TS brain animal model.
  • HSV-T0 ⁇ Hex Herpes simplex vector encoding for the hexosaminidase (Hex) A alfa-subunit
  • HSV-T0 ⁇ Hex Herpes simplex vector encoding for the hexosaminidase (Hex) A alfa-subunit
  • replication-deficient HSV whose DNA encodes hexosaminidase (Hex) A, preferably the alfa-subunit (HSV-TO ⁇ Hex), and a method of expressing Hex A in a subject, preferably in the subject's brain, preferably by administration of said HSV, preferably so that the HSV is delivered to the internal capsule.
  • HSV hexosaminidase
  • HSV-TO ⁇ Hex alfa-subunit
  • TS neurodegenerative lysosomal Tay-Sachs
  • Hex Hexosaminidase
  • HSV- TOalphaHex Herpes simplex vector encoding for the Hex A alpha-subunit
  • the anatomic structure of the brain internal capsule to optimally distribute the missing enzyme.
  • HSV- TOalphaHex Herpes simplex vector encoding for the Hex A alpha-subunit
  • GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in one month of treatment.
  • NTFs Neurotrophic factors
  • HSV-l replication-deficient herpes simplex virus- 1
  • fibroblast growth factor FGF-2
  • CNTF ciliary neurotrophic factor
  • BDNF brain derived neurotrophic factor
  • M&C FOLIO WPP896 ⁇ 58 P( ⁇ EP 2005/003639 22 (GDNF) alone or in combinations. Therefore, according to a further aspect of the present invention, there is provided replication-deficient HSV whose DNA encodes at least one neurotrophic factor (NTF), and a method of expressing the NTF in a subject comprising administering said HSV.
  • NTF neurotrophic factor
  • HSV replication-deficient HSV, as described herein, comprising DNA encoding at least one Neurotrophic Factor (NTF) in medicine.
  • NTF Neurotrophic Factor
  • TDD Tay-Sachs Disease
  • NTF nerve-growth factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glial-derived neurotrophic factor
  • the NTF is FGF-2 or BDNF.
  • synerg occurs between different NTFs, particularly between FGF-2 and BDNF.
  • FGF-2 and BDNF are encoded by the HSV.
  • Example 1 replication-deficient HSV vectors in treatment of intracelleular bacterial infection
  • MHC Major histocompatibility complex
  • CD8 + T cells recognizing antigenic peptides derived from pathogens play major roles in protection against intracellular bacteria like Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes (for a review, see references 15 and 16).
  • Vaccines utilizing inactivated or recombinant bacteria have been demonstrated to elicit both CD4- and CD8-T-cell activation (16), but they seem to be inefficient stimulators of effector (25) and memory (34) T cells if compared side by side with live bacteria (25) or recombinant viral vaccines (34). In this respect, inactivated intracellular bacterial vaccines are similar to natural M.
  • tuberculosis (44) and L. monocytogenes (33) infections which fail to induce sufficient immunological memory to prevent recurrent infections.
  • DNA vaccines have been demonstrated to induce protection against infections with M tuberculosis (43) and L. monocytogenes (9).
  • DNA vaccines with attenuated bacteria (12), protein antigen (42), or modified vaccinia virus (28) in heterologous prime-boost vaccination protocols could further optimise protection.
  • DNA vaccines alone are not sufficient to induce maximal protective immunity and, in a clinical setting, might require complex vaccination arrangements.
  • recombinant vaccinia virus could elicit only limited protection against intracellular bacteria and delayed, rather than prevented death after infection (2).
  • efficacious "single-shot" vaccines to intracellular infection have yet to be developed.
  • CD8 T cells are the effector cells of choice for intracellular.
  • Replication-deficient (10, 30) or disabled infection with single-cycle he ⁇ es simplex virus (HSV) (3, 27) has been shown to induce strong irnrnune responses against HSV-derived antigens.
  • rHSV-1 replication-defective HSV-1
  • OVA ovalbumin
  • FIG. 1 Visualization of antigen-specific CTL expansion after vaccination.
  • PBL were stained with anti-CD8-APC and H " 2K /OVA257-264 tetramers-phycoerythrin and analysed by flow cytometry before (a) and 7 days after vaccination with pcDNA3-OVA (b), T0H-OVA (c), or T0-GFP (4 x 10 6 virus particles i.v.) (d).
  • FIG. 2 Protection against L.m.-OVA infection,
  • C57BL/6 mice were left ummmunised or vaccinated with pcDNA3-OVA, T0H-OVA, or T0-GFP (4 x 10 6 virus particles i.v.) (day 0) and infected i.v. with 5 x 10 4 (two times the LD 50 ) L.m.-OVA cells 8 days later.
  • mice At day 10 postinfection (day 18 postimmunisation), spleens of surviving mice were analysed for presence of Tet + CTL (d).
  • the bar graphs (b and d) depict the percentage (mean +/- standard deviation [error bars]) of Tet- positive cells among total CD8 + T cells (five mice/group), (e)
  • As a control for the antigen-specificity of protection vaccinated mice were infected with 5 x 10 4 wild-type L. monocytogenes cells (not expressing OVA), and survival was monitored. Abbreviations: no imm, no immunization; p.i., postinfection
  • FIG. 3 Protection against infection with high dose of L.m.-OVA.
  • C57BL/6 mice were vaccinated with T0H-OVA, TO-GFP (4 x 10 6 virus particles i.v.), or ⁇ cDNA3-OVA and infected with 10 5 (four times the LD50) L.m.-OVA cells 7 days postvaccination.
  • FIG. 4 Long-term protection against L.m.-OVA infection,
  • FIG. 5 Monitoring of OVA-specific CD4 + -T-cell responses in vivo following rHSV-1 vaccination.
  • BALB/c mice received 2.5 x 10 6 na ⁇ ve DOl l.lO cells at day -1 and were immunized with gene gun (pcDNA3-OVA), T0H-OVA, or TO-GFP (4 x 10 6 virus particles) i.v. at day 0.
  • the frequency of DOl l.lO T cells present in the inguinal lymph nodes and spleen was measured by flow cytometry.
  • a-c) Cell suspensions were stained with anti-CD4, and KJl-26-fluorescein isothiocyanate (DOll.lO TCR-specific).
  • the percentages of CD4 + /KJ126 + cells in lymph nodes of control (TO-GFP)-, pcDNA3-OVA-, and TOH-OVA-immunized mice at day 5 postimmunization are indicated in the dot plots.
  • Kinetics of DOll.lO T-cell expansion is shown for lymph nodes (d) and spleen (e).
  • d and e the total cell numbers +/- standard deviations (error bars) of CD4 + KJ1- 26 + cells are indicated (three to four animals per group).
  • Sera were obtained from mice immunized with pcDNA3-OVA, T0H-OVA, or TO-GFP (4 x 10 6 virus particles i.v.) at the indicated time points postvaccination, and OVA-specific antibody serum levels were determined by enzyme-linked immunosorbent assay. Preimmune serum (day 0) from each group was determined with a pool of sera. Results are expressed as the mean of optical density at 450 nm (O.D. 450 nm) +/- standard deviation (error bars) from at least three individual mice per group.
  • the efficiency of a vaccine is directly proportional to its capacity to activate T cells and to generate a memory T-cell pool.
  • Development of memory T cells has been shown to be directly proportional to the intensity of the primary response (31), and in several viral infection models, the viral dose correlated positively with T-cell memory development (23, 34, 35).
  • Recombinant replication- deficient mutant HSV-1 not only induced strong expansion of CD8 + CTL (Fig. 1), it also proved to be an extremely efficient vaccine against infection with an intracellular bacterium.
  • TOH-OVA unlike DNA vaccination, did not induce significant CD4 T helper cell or antibody responses specific for the recombinant antigen. This may be irrelevant, as protection against intracellular bacterial pathogens such as L. monocytogenes is largely CTL mediated (17, 39). Although antibody responses could be important to neutralize L. monocytogenes immediately after bacterial entry into the host and might therefore be considered a prerequisite for the establishment of an efficient long-term vaccination effect against intracellular bacteria (8), a lack of pathogen-specific antibodies did not negatively affect the ability of HSV-1 -derived vaccines to provide long-term memory protection in our system.
  • HSV-1 -derived vectors are not affected by pre-existing immunity to HSV-1 (4). In the light of 75% of the adult human population have been previously exposed to HSV-1, this is an important prerequisite for the efficient usage of these vaccines (13).
  • HSV-1 -derived vectors induce strong CTL responses, making them a promising candidate for vaccines against intracellular " bacterial infection.
  • mice All mice were bred and maintained under standard conditions in the animal facilities of the Institute for Immunology, Ludwig-Maximilians-University Kunststoff; the Institute for Microbiology, Immunology and Hygiene, Technical University Kunststoff; or the Department of Pharmacy, University of Ferrara. DOll.lO mice (expressing transgenic T-cell receptors [TCR] specific for OVA323-339/MHC class II I-A d ) were obtained from Jackson Laboratory, Bar Harbor, Maine.
  • TCR transgenic T-cell receptors
  • a BamHiyXhoI fragment of rabbit ⁇ globin was cloned into a BamHI/XhoI-opened pcDNA3 vector (Invitrogen) to create pcDNA3- ⁇ globin.
  • the pcDNA3-OVA vector encoding the secreted form of chicken ovalbumin (OVA) was constructed by cloning a 1.9-kb EcoRI fragment from the plasmid pAc-Neo-OVA (provided by F. Carbone, Melbourne, WEHI, Australia), which contained the entire coding sequence of OVA, into the EcoRI site of pcDNA3- ⁇ globin.
  • Plasmids were prepared from Escherichia coli with Qiagen (Hilden, Germany) Mega Kits.
  • a recombination plasmid (pB410H:OVA) was constructed by introduction of HCMV- ⁇ globin OVA cDNA expression cassette into the U141 locus of HSV-1.
  • the cDNA under the transcriptional confrol of the human Cytomegalovirus promoter was inserted in a Smal/Xbal-opened pBBSK plasmid between the two UL41 fragments (map positions 93,858 to 92,230 and 91,631 to 90,145) 100 bp downstream of the HSV immediate-early ICPO promoter.
  • This plasmid (pB410H:OVA) was recombined with the genome of TOZGFP using the previously described Pac-facilitated lacZ substitution method (19).
  • TOZGFP is a nonreplication HSV viral vector background that has low toxicity due to the deletion in three immediate early genes (ICP4 and ICP27, which are essential for viral replication, and ICP22) with cDNA encoding GFP inserted into the ICP22 locus and an insertion of LacZ in the UL41 locus.
  • the recombination was carried out using standard calcium phosphate fransfection of 5 ⁇ g of viral DNA and 1 ⁇ g of linear recombination plasmid pB410H:OVA.
  • Transfection and isolation of the recombinant virus was performed in 7b Vero cells (African green monkey kidney cells CCL81; American Type Culture Collection, Manassas, Va.) capable of providing the essential ICP4 and ICP27 HSV gene products.
  • the recombinant virus containing the OVA cDNA was identified by isolation of a clear plaque phenotype after X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ - D-galactopyranoside) staining.
  • TOH-OVA virus was purified by three rounds of limiting dilution and the presence of the fransgene was verified by Southern blot analysis.
  • Viral stocks of the TOH- OVA virus and the control vector TO-GFP (derived from TOZGFP without lacZ reporter gene in UL41 locus) were prepared and titrated using 7b cells.
  • DOll.lO cells were prepared from lymph nodes and spleens of transgenic mice. Briefly, spleen and lymph nodes were taken out, and single-cell suspensions were prepared. Erythrocytes were removed by osmotic lysis, and after determining the percentage of DOll.lO TCR-trahsgenic T cells by flow cytometry, 2.5 x 10 6 transgenic T cells were injected intravenously into the recipient mice.
  • Naked DNA immunization was performed by gene gun administration (Bio-Rad Laboratories, Hercules, Calif.). Cartridges of DNA-coated gold particles were prepared according to the manufacturer's instructions. For each preparation, gold particles (25 mg; diameter, 1 ⁇ m) were coated with 200 ⁇ g of DNA. Mice were anaesthetized prior to vaccination with a mixture of Ketavet/Rompun in phosphate-buffered saline (PBS). A total of 8 ⁇ g of plasmid DNA was delivered to the shaved abdominal skin of adult mice with a discharge pressure of 400 lb/in 2 . For TOH-OVA vaccination, frozen virus stocks were thawed on ice, diluted in PBS to 4 x 10 6 T0H- OV A/200 ⁇ l, and injected intravenously (i.v.).
  • PBS phosphate-buffered saline
  • Enzyme-linked immunosorbent assay For the detection of OVA-specific antibodies, 96-well microtiter plates (Nunc Maxiso ⁇ , Nunc, Wiesbaden, Germany) were coated with OVA (15 ⁇ g ml; Sigma Chemical Co., St. Louis, Mo.) at room temperature overnight. Plates were blocked (PBS, 0.5% milk powder, 0.05% NaN3), and immune sera (diluted 1:100 in blocking buffer) were incubated for 2 h at room temperature.
  • Lymphocytes were analysed using the following monoclonal antibodies (MAbs): anti-CD4- PerCP (L3T4), anti-CD8a-PerCP (Ly2), and anti-CD62L (Mel-14) from PharMingen (San Diego, Calif.) and KJl-26-fluorescein isothiocyanate specific for DOll.lO TCR, anti-CD8a- APC (Ly2), and anti-CD44-PE from Caltag (Burlingame, Calif). Biotinylated MAbs were detected with streptavidin-APC (Caltag).
  • Tetrameric complexes were stored at 2 mg/ml at 4°C in PBS (pH 8.0) containing 0.03% sodium azide, pepstatin (1 ⁇ g/ml), leupeptin (1 ⁇ g/ml), and 1 mM EDTA. The reagents were frequently tested on antigen-specific T-cell lines to document staining quality.
  • mice were infected i.v. with L. monocytogenes expressing the secreted form of OVA (36) (L.m.- OVA, kindly provided by Hao Chen, Philadelphia, Pa.). Viable bacterial counts within spleen and liver were determined by homogenizing the respective tissue in PBS containing 0.05% Triton X- 100 and plating on brain heart infusion agar plates (Life Technologies, Gaithersburg, Md.). L. monocytogenes colonies were identified by their characteristic mo ⁇ hology and by Gram staining. RESULTS
  • Recombinant HSV-1 vaccination protects against L. monocytogenes infection.
  • HSV-1 vaccines Protection from infection with a high dose of X. monocytogenes is induced by HSV-1 vaccines, but not by DNA vaccination.
  • TOH-OVA-derived vaccines induce potent long-term protection against infection with the facultative intracellular bacterium L. monocytogenes and generate a large memory CTL pool capable of clearing even large bacterial loads very efficiently.
  • gene gun vaccination induces lower frequencies of specific CTL sufficient for the protection of mice from challenge with lower but lethal L isteria-dosss but are less efficient at achieving complete bacterial clearance.
  • mice that had previously received adoptively transferred TCR-transgenic OVA-specific DOl l.lO CD4 + T cells (18).
  • DOl l.lO transgenic T cells recognize the OVA323-339 peptide in the context of MHC class II I-A d and can be detected with the clonotypic TCR-specific MAb KJ1-26 facilitating the monitoring of their activation and expansion following vaccination with the specific antigen.
  • control vaccinated mice contained very few CD4 + KJ1-26 + T cells (TO-GFP) (Fig. 5a and d), the percentages increased 5- to 10-fold and total numbers increased 10- to 30-fold in the draining lymph nodes in gene gun- vaccinated animals (Fig. 5b and d) 5 days after vaccination.
  • HSV-1 -derived vaccines are weak inducers of CD4 T helper and antibody responses but activate a large and efficient CD8 + T-cell response.
  • HSV-2 he ⁇ es simplex virus type 2
  • a cytosolic he ⁇ es simplex virus protein inhibits antigen presentation to CD8 ⁇ T lymphocytes. Cell 77:525-535.
  • Example 2 Replication-deficient HSV comprising angiogenic inhibitors. cytokines and suicide genes for use in Anticancer treatments
  • HSV vectors are created containing mIL12 or mGM-CSF provided by Invivogene. Production and secretion of cytokines molecules following viral infection of various cell lines will be confirmed by western blot and the quantification will be performed by specific ELISA kits.
  • the vectors are generated by inserting the transgene expression cassettes, in which the cytokine genes are under the transcriptional control of the strong human cytomegalovirus immediate early promoter (HCMV), into the Usl locus of HSV genome by homologous recombination with the method previously described (Krisky D, Marconi et al. 1997).
  • HCMV cytomegalovirus immediate early promoter
  • TL12 (frivivogen) or GM-CSF sequences will be introduced in the same viral locus to avoid differences in gene expression due to promoters and viral location.
  • THZ-1 is a recombinant virus deleted in the ICP4, ICP27 and ICP22 immediate early genes with the lacZ reporter gene under HCMV promoter, in the ICP22 locus.
  • THZ-1 viral DNA and the recombinant plasmids containing the cytokine genes are co-transfected into a complementing cell line (7b), which provides, in trans, the essential viral genes ICP4 and ICP27.
  • the gene expression cassettes are introduced into the background of the vector THZ.5, which is a mutant deleted in the ICP4, ICP27 and ICP22 immediate early genes and in the non-essential gene UL13 of HSV genome that contains LacZ reporter gene under HCMV promoter (THZ.5).
  • This vector (T-MMP9GM-CSF) as well as all the others in this proposal is based on a recombination methodology developed in J.C. Glorioso's laboratory, which results in high frequency recombinants (22, 28). The reason for the construction of these two last vectors is to have their expression cassettes in a different viral location from IL12 insertion locus.
  • a furhter vector which carries both cytokine genes GMCSF and/or IL12 (THGM-CSF/IL12 or T-MMP9GM-CSF/IL12).
  • GMCSF cytokine genes
  • IL12 IL12
  • GM-CSF is expressed under tumor-specific promoters in order to increase vector expression only in targeted tumor cells and avoid the toxicity induced by an over-expression of this cytokine.
  • the cytokine expression of all the above described vectors is detected and measured by ELISA.
  • the vectors expressing mIL12 or mGM-CSF are combined with vectors, constructed in the previous project and expressing the angiostatic fusion protein, Endostatin- Angiostatin or Endostatin-Kringle5, in different ways.
  • production and secretion of the antiangiogenic and cytokines molecules following viral infection of various cell lines is confirmed by western blot and the cytotoxic activity of TK will be evaluated in presence of GCV with standard MTT assay.
  • All these recombinant viruses have also their own HSV-TK gene where the TK endogenous promoter has been substituted with the HSV immediate early promoter ICP4 in order to ensure the expression of this suicide gene in a replication-defective HSV mutant.
  • We will identify the recombinants by screening progeny viruses by Southern blot analysis for the transgenes sequences. Positive isolates are purified following three rounds of limiting dilution and the protein expression will be determined by Western blot analysis and the cytokine expression will be detected and measured by ELISA. Construction of HSV vectors co-expressing cytokine, suicide genes and Cx43.
  • the vectors expressing IL12 or GM-CSF are combined with vectors expressing TK and Cx 43 gene.
  • the tumor cell lines are infected with the cytokine vectors and the secreted proteins quantified by ELISA kits.
  • the cytotoxic activity of TK is evaluated in presence of GCV with standard MTT assay.
  • the THIL12 or THGM-CSF vectors are genetically crossed with the mutant virus that carries Cx43 in the UL41 locus (29) to create T0CX-IL12 or TOCX-GMCSF. These mutants have their natural TK under the ICP4 promoter. Progeny viruses are then screened for the presence of the transgenes by Southern blot analysis and the protein expression identified by Western blot analysis. Cytokine expression is identified by ELISA.
  • Tumor cells are infected with the control vector (THZ4) and with the vectors expressing TK or TK and Cx43.
  • the cells are infected at different MOIs and plated in 96well plates in presence or absence of GCV. At 24h intervals the cell viability is determined by MTT assay. The results are plotted as the percentage of survival relative to mockinfected cells.
  • ECV304 and primary endothelial cell lines HUVEC are treated with the recombinant basic fibroblast growth factor (rbFGF) and vascular endothelial growth factor (rVEGF) in order to induce angiogenesis.
  • rbFGF basic fibroblast growth factor
  • rVEGF vascular endothelial growth factor
  • different tumor cell lines are infected with vectors expressing anti-angiogenic factors in different combinations and tested their antiproliferative and antimigratory activity on ECV304 and on HUNEC cells, using MTT-based assays, conventional migration chambers and specific tube formation assays.
  • ⁇ on-replicative HSV-1 based vectors are able to express biologically active angiogenesis inhibitors and suicide genes.
  • Anti-proliferative activity in vitro is used to evaluate the biological activity of the recombinant proteins.
  • MTT assay Proliferation of endothelial cells by MTT assay, which determines the metabolic activity of mitochondria and correlates well with the number of viable cells.
  • Media from LLC, B16 and GL- 216 (2*10 ⁇ ) cells infected with the recombinant vectors at MOI of 2 is collected and overlaid on ECV304 or HUVEC.
  • bFGF 3ngr/ml is added as angiogenic stimulus.
  • Negative controls are represented by ECV304 or HUVEC cells freated with medium obtained from non-infected tumor cells or infected with a control vector (THZ1 or THZ5). After an incubation of 5 days, ECV304 growth or HUVEC is determined by a colorimetric, tetrazolium-based (MTT) assay.
  • MTT colorimetric, tetrazolium-based
  • Results are expressed as percentage of cell migration, compared with migration induced by conditioned media from uninfected tumor cells; 10 Qgr/ml of recombinant human antiangiogenic proteins (angiostatin and endostatin) is used as a positive control.
  • the endothelial tube formation is evaluated and visualized through a commercial kit.
  • the cells a mixture of fibroblasts and endothelial cells
  • the cells are infected with the vectors (MOI 1) at days 1, 4, 7 and 10 post cells seeding.
  • Positive kit confrol are treated with VEGF 2 ngr/ml; negative control are treated with suramin 20 mM or the viral vector control.
  • the cells are incubated with anti-CD31 antibody, in order to evidence and to count the formed tubes.
  • Microvessel mo ⁇ hology, neovascularization and infiltrating cell phenotypes are analyzed on tumor tissue sections using immunohistochemical techniques.
  • the tumors implanted in the right flank will allow us to test the efficacy of the different recombinant vectors by monitoring the tumor mass growth with calipers over the course of the experiments.
  • To determine the number of cytokine secreting cells spleens and tumors are dissected and the cytokine in situ production is carried out by ELISPOT method. If these vectors are efficient, the tumor volume decreases and the immune response against the tumor is increased.
  • Murine Lewis lung carcinoma (LLC), B16 melanoma and GL-261 glioma cells are implanted in the right flank of 7 weeks old C57BL/6 mice to directly test the efficacy of angiostatic genes, cytokines and TK suicide gene following prodrug adminisfration and to analyze the anti-tumor treatment response related to the tumor model.
  • tumors are treated with PBS solution and vectors without therapeutic genes as negative control and with recombinant vectors expressing cytokines molecules alone and/or in combination with anti-angiogenic and suicide genes.
  • the group of mice treated with combined therapy is compared with animals inoculated with vectors expressing only angiostatic molecules or only cytokines or TK/GCV.
  • Physiologic solution and pro-drugs are administered intraperitoneally and treatment will start when tumors are palpable. Tumor volumes are measured with a digital calliper every two days; each time point will represent the average of 6 mice in each group.
  • mice are sacrificed at different times post tumor cells injection. Histologic analysis is done on tumor sections to define, microvessel mo ⁇ hology and neovascularization and tumor infilfrates using immunohistochemical techniques. Furthermore to determine also the number of cytokine secreting cells, spleens and tumors are dissected and the cytokine released will be carried out by ELISPOT method. The end point to use three different tumor cell lines is to test this multifactorial therapy and to verify the susceptibility of the different tumors to respond to these angiostatic, cytokines and TK molecules and to analyze if their synergistic effect is equally efficient in eradicating different neoplasies.
  • TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer.
  • Hess, S. D., N. K Egilmez, N. Bailey, T. M. Anderson, E. Mathiowitz, S. H. Bernstein, and R. B. Banker! 2003.
  • Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma.
  • J Immunol 170:400-12. 19. Jia, S., F. Zhu, H. Li, F. He, and R. Xiu. 2000. Anticancer freatment of endostatin gene therapy by targeting tumor neovasculature in C57/BL mice. Clin Hemorheol Microcirc 23 :251-7. 20. Ka ⁇ off, H. M., D. Kooby, M. D'Angelica, J. Mack, D. H. Presky, M. D. Brownlee, H. Federoff, and Y. Fong.
  • Example 3 HSV-based vectors for the treatment of HIV-associated tumours, such as neoplasias
  • angiostatic factors promoting tumor regression along with enzyme-directed prodrug activation (tumor suicide) and/or IL-12 cytokine.
  • a replication-defective HSV vector which expresses a fusion anti-angiogenic molecule, such as angiostatin and endostatin and
  • Both vectors have TK suicide gene that has been shown to improve the efficiency of anti-tumor gene therapy. Furthermore, new therapeutic combinations are accomplished by coupling, in the same vectors, the genes with angiostatic modulatory effect with the cytokine IL12 gene, which has a strongly immunomodulatory properties (40, 66). 11-12 promotes the proliferation of T cells, NEC cells and tumor-infiltrating lymphocytes (TIL cells), in addition, it can induce a cascade of other cytokines and chemokines which possesses significant antiangiogenic properties (17, 59).
  • HCMV human cytomegalovirus immediate early promoter
  • HSV ICP4 immediate early promoter HSV ICP4 immediate early promoter
  • the vectors are tested, in vivo, in appropriate animal models.
  • the BDF and nude Balb/c mice are inoculated with T53 BKV/Tat derived tumor cell line and subsequently the tumor mass are treated with the vectors expressing: endostatin/angiostatin, endostatin/kringle genes, or both recombinant vectors, with or without addition of GCV.
  • the mice are treated in order to inhibit tumor growth and reduce its mass.
  • HIV-1 tat acts as a growth factor and induces angiogenic activity in BK virus/tat transgenic mice. Antibiot Chemother 46:88-101.
  • HIV type 1 extracellular Tat protein stimulates growth and protects cells of BK virus/tat transgenic mice from apoptosis. AIDS Res Hum Retroviruses 11:1039- 48.
  • Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 272:22924-8.
  • IFN-gamma induces endothelial cells to proliferate and to invade the extracellular matrix in response to the HIV-1 Tat protein: implications for ATDS-Kaposi's sarcoma pathogenesis. J Immunol 162:1165-70.
  • Kaposi's sarcoma cells of different etiologic origins respond to HIV-Tat through the Flk-1/KDR (VEGFR-2): relevance in AIDS-KS pathology. Biochem Biophys Res Commun 273:267-71.
  • Endostatin an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277-85.
  • Angiostatin a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth. Cold Spring Harb Symp Quant Biol 59:471-82.
  • IFN-gamma-inducible 5 protein- 10 is essential for the generation of a protective tumor-specific CD8 T cell response induced by single-chain IL-12 gene therapy. J Immunol 166:6944-51.
  • a critical issue in the field of vaccine development is to have suitable animal models to test the safety, irnmunogenicity and efficacy of a vaccine formulation.
  • the present challenge model is based on recombinant replication-competent HSV-1 or HSV-2 expressing the vaccine antigen, e.g. HIV antigens.
  • the method is generally useful in pre-clinical vaccine research, for example, in investigations aimed at studying the efficacy of any vaccine candidate.
  • Safety, immunogenicity and protection experiments will be performed in animal models (e.g murine) that may be of outmost relevance to speed up vaccine candidate testing and transition to clinical trials.
  • the antigen is introduced in a HSV locus which is not essential for HSV-1 and HSV-2 replication (e.g. UL41, Us4, Us5) (Marconi et al., Proc. Natl. Acad. Sci. USA, 1996).
  • HSV-1 and HSV-2 replication e.g. UL41, Us4, Us5
  • recombinant viruses are able to replicate and to induce disease and death in mice after inoculation of a lethal dose by systemic and mucosal routes. Survival of immunized mice 10-15 days after challenge with a lethal dose of recombinant HSV-1 expressing the vaccine antigens is the efficacy endpoint.
  • the vaccine antigen e.g. HIV-1 genes [t ⁇ t, gag (clade B), and env (clades A, B, and C)] is introduced in the UL41 locus of the HSV-1.
  • the vaccine antigen may be introduced in the Us5 locus of HSV-1.
  • HSV-based challenge system is based on HSV-2, or genital he ⁇ es, which is more pathogenic in the genital tract as compared to HSV-1.
  • Balb-c mice inoculated in the vagina or in the footpad are highly susceptible to HSV-2 infection.
  • HSV-2 infection Although both wild-type HSV-1 and HSV-2 infect and induce disease in mice after infra- vaginal inoculation, the lethal dose of HSV-1 is 1-2 logs higher (depending on the strains) than that of HSV-2.
  • replication competent HSV-2 viruses expressing the vaccine antigens may he more efficacious as a vaginal challenge model, as compared to recombinant HSV-1.
  • the HSV virus carrying a deletion in a non-essential gene, such as UL41, Us4 or Us5 locus is be generated by homologous recombination of the HSN-1 or HSV-2 genome with a plasmid carrying the LacZ gene flanked by HSV-1 or HSV-2 sequences.
  • the deleted virus is then used to create the recombinant HSV-1 or HSV-2 viruses carrying the e.g. HlV-vaccine antigens in the deleted locus.
  • the new recombinant HSN-1 or HSV-2 viruses are analyzed by Southern blot and PCR, and tested for expression of the transgenes e.g. by immunofluorescence and western blot.
  • LDL 00 lethal dose following systemic and mucosal (i.e intra- vaginal, intra- rectal) inoculation.
  • Vero African green monkey kidney
  • Balb-c mouse cells were maintained in Dulbecco's modified minimal essential medium (BioWhittaker) supplemented with 5% fetal bovine serum, 1% Glutamine and 1% penicillin/streptomycin.
  • Escherichia coli (Stratagene) strain DH5 ⁇ was used in plasmid cloning procedures. Bacteria were grown in Luria-Bertani medium (for liquid culture) or in Luria-Bertani agar plates, both supplemented with antibiotics as appropriate (Ampicillin 100 ⁇ g/ml or Kanamycin 50 ⁇ g/ml).
  • Plasmids. pET28a-modi-Nef plasmid was kindly provided by V. Erfle (GSF, Kunststoff, Germany), pKCMV-p37 (Gag p24 + pl7) and pKCMV-gpl60 (Env clade A, B, C separately) by B. Wahren (Karolinska Institutet, Stockolm, Sweden).
  • p41plasmid (a pBlueScript comprising the HSV-UL41 flanking regions), alone and with the expression cassette containing LacZ gene has been previously described (Krisky DM et al, 1997).
  • pcDNA 3.1 " plasmid was purchased from Invitrogen, pTZ18U plasmid from Sigma.
  • mice Balb-c female mice (5-6 weeks old) were purchased from Charles River (Milan, Italy) Replication competent viruses expressing LacZ.
  • the virus HSV-1 ⁇ 41 or HSV-2 ⁇ 41-1 were created by insertion of a deletion in the UL41 locus of the HSV-1 and HSV-2 genome according to the method previously described (Krisky DM et al, 1997). Briefly, a plasmid containing the HSV ICPO-immediated early promoter driving the expression of the lacZ reporter gene (transgene cassette), cloned into the UL41 locus of HSV (Pad restriction endonuclease site), was constructed.
  • the transgene cassete is flanked by HSV-1 or HSV-2 UL41 viral sequenses.
  • the recombinant virus was created by homologous recombinatio between the palsmid and the HSV genome.
  • Vero cells were co-transfected with the plasmid and either the HSV-1 wild-type or HSV-2 wild-type genome DNA.
  • the recombinant virus carrying the deletion in the UL41 locus were selected and isolated based on lacZ gene expression, according to standard procedures (Krisky DM et al, 1997 ). The correct insertion was confirmed by Southern blot analysis.
  • HSV-l/Us5 ⁇ -l or HSV-2/Us5 ⁇ -l and HSV-2/Us4 ⁇ -2 with a deletion in the Us5 locus of HSV-1 and HSV-2, corresponding to glycoprotein JI (gJl), or in the Us4 locus of HSV-2, corresponding to glycoprotein G (gG).
  • HCMV human cytomegalovirus
  • HCMV human cytomegalovirus
  • This plasmid was co-transfected in Vero cells either with LV-HSV-1 wild-type strain or G- HSV-2 wild-type strain.
  • Plasmid constructs In order to have the genes of interest under the control of the HCMV promoter and between the UL41 flanking regions, two cloning steps were necessary. For construction of plasmids p41-Tat, p41-Nef, p41-Gag, p41-EnvA, p41-EnvB and p41-EnvC, the following procedure was carried out. The tat gene was excided from original plasmid pCV-tat (Arya et al., Science 1985) with Pstl and then ligated into the Pstl site of plasmid pTZ18U (Sigma) to generate the intermediate expression plasmids pTZ18U-Tat.
  • Plasmid pB410-tat was then constructed by introduction of the HIV-1 tat cDNA (350 pb) from pTZ18U-Tat into the UL41 HSV1 sequences (HSV genomic positions 90.145-91.631 and 92.230-93.858) of plasmid pB41, which has been described elsewhere (Krisky DM et al, 1997), under the transcriptional control of the HSV1 immediate-early ICPO promoter.
  • nef gene was excided from plasmid pET28a-modi-Nef with Xbal/EcoRI, while the other genes (gag and env) were excided with Sall/EcoRI from pKCMV-p37 and pKCMV-gpl60, respectively.
  • the fragments were then ligated into the Xbal/EcoRI (nef) or XhoI/EcoRI (gag and env) site of pcDNA 3.1 " (InVitrogen) to generate intermediate expression plasmids (pcDNA3.1- Nef, pcDNA3.1-Gag, pcDNA3.1-EnvA, pcDNA3.1-EnvB and pcDNA3.1-EnvC) where the nef, gag and env genes are under the transcriptional control of the HCMV-immediate early promoter.
  • Fragments with HIN genes under HCMV promoter were then cleaved from the pcD ⁇ A3.1- derived plasmids with NruI/EcoRI and ligated into the Sma ⁇ coRI sites of the pB41 plasmid.
  • HSV-1 recombinant vectors expressing HIV-1 proteins HSV-1 recombinant vectors expressing HIV-1 proteins.
  • LV-LacZ viral genome was isolated from infected Vero cell lysates by the proteinase K / phenol-chloroform extraction procedure (Sambrook J. et al, 1989). Cotransfections of LV-LacZ viral DNA, cleaved with Pad (in order to excide Lac Z), and the above described plasmids containing the HIV genes, linearized with Notl, were performed in Vero cells using calcium phosphate precipitation technique (Knipe DN et al, 1979), then in vitro titer was assessed with the methyl-cellulose method as previously described (Fendrick JL et al, 1983). After each co-fransfection, recombination percentage was evaluated by using LacZ as a reporter gene.
  • Plaque purification was carried out by 3-4 Limiting Dilution (LD) rounds (Krisky et al. 1997), each followed by a Southern-Blot (SB) or Dot Blot (DB) analysis to confirm the presence of the transgenes and their correct insertion into the viral genome.
  • LD Limiting Dilution
  • SB Southern-Blot
  • DB Dot Blot
  • Vero and Balb-c cells were infected with 1 multiplicity of infection (m.o.i.) of recombinant virus, harvested at different time points and diluted in loading buffer containing protease inhibitors and dithiothreitol (DTT).
  • Cell extracts corresponding to 10 ⁇ g of total proteins, were loaded onto 12% SDS-polyacrylamide gel and analyzed by western blot using specific monoclonal antibodies (ARP 3061 1:1000 for Gag and EVA 307 1:250 for EnvC) and a goat-anti-mouse IgG antibody HRP-conjugate (Sigma) (1:2500).
  • Antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 1 with HSV-1 wild-type virus.
  • Recombinant proteins and MAbs were obtained from the Centralized Facility for AIDS Reagents (EVA Program), UK. Large-scale virus production. In order to have a stock of serum-free purified virus, cells (24 to 32 150 mm 2 tissue culture flasks) were infected with 0.02 multiplicities of each virus and harvested once the infection was well spread (approximately 24 hours post-infection).
  • the recombinant HSV viruses were purified with Optiprep® gradient (Axis-Shield) according to the manufacturer's instructions, diluted in PBS, divided in 30-100 ⁇ l aliquots and stored at - 80°C.
  • the in vitro titer was assessed with the methyl-cellulose method as previously described (Fendrick JL et al, 1983).
  • Vaginal challenge One week before challenge, female Balb-c mice are treated with 2 mg/100 ⁇ l of Depo-Provera ® (Depo-medroxy-progesterone acetate; Pharmacia & Upjohn) given subcutaneously in the neck. This drug brings the animals at the same estrous stage, minimizing infection variability (Kaushic C et al, 2003).
  • Depo-Provera ® Depo-medroxy-progesterone acetate; Pharmacia & Upjohn
  • This drug brings the animals at the same estrous stage, minimizing infection variability (Kaushic C et al, 2003).
  • the day of the challenge the virus is thawed in ice, sonicated for 5 seconds, and briefly stored on ice.
  • Mice are anaesthetized with 5% isoflurane to allow scraping of the vagina with a pipe scraper (in order to remove the mucus that could trap the virus) and then inoculated with the purified virus using a pipette-tip. Onset and follow-up of the infection is then observed for at least 15 days.
  • Recombinant HSV-1 viruses carrying HIV-1 genes [t ⁇ t, gag, env (clade B) and env of clade A and C] in the UL41 locus, namely LV-Tat, LV-Gag, LV-EnvA, LV-EnvB, LV-EnvC ( Figure 7) were generated and characterized by Southern blot analysis, as described in the materials and methods section. Expression of HIV-1 proteins was assessed following infection of Vero cells or Balb/c fibroblasts with the HSV-1 recombinant viruses by western blot, 24 and 48 hours post- infection. Controls were represented by cells infected with the LV-lacZ vector or non-infected cells.
  • mice Female Balb-c mice are more susceptible to HSN infection as compared to C57BL6 . mice (Lundberg P et al, 2003), thus all experiments were carried out in Balb-c mice.
  • mice were inoculated with 1.4 x 10 6 , 1.4 x 10 7 , 1.4 x 10 8 pfu of LV-Tat infraperitoneally (in 100 ⁇ l of physiologic solution/mouse) or intravaginally (10 ⁇ l mouse).
  • This drug transforms a proliferative endometrium into a secretive endometrium, by inhibiting hypophysis in gonadofropin secretion and then hampering follicular maturation and ovulation.
  • Inoculation of Depo-Provera ® brings the mice at the same estrous stage and render them more susceptible to HSV infection (Kaushic C et al, 2003).
  • mice were infected infra- vaginally with 1.5 x 10 8 pfu/10 ⁇ l, 3 x 10 s pfu/20 ⁇ l, 6 x 10 8 pfu/40 ⁇ l, from a viral stock that had a titer of 1.5 x 10 10 pfu/rnl.
  • Figure 9 A shows that LV-Tat killed 9/10 of the mice at the dose of 3 x 10 8 pfu.
  • the reason why a lower mortality was observed with the dose of 6 x 10 8 pfu may depend on a technical problem, namely that to infect with the dose of 6 x 10 it was necessary inoculate 40 ⁇ l of viral solution.
  • mice were divided into three groups and inoculated mfra- vaginally. One group was inoculated with 3 x 10 pfu/10 ⁇ l of LV- Tat at day 1, the second group was inoculated with same dose both at day 1 and at day 2, and the third group received the same virus dose at days 1, 2 and 3. Mice were then observed daily for disease onset and death.
  • mice were inoculate infra-vaginally with the same dose (1.35 x 10 10 pfu/10 ⁇ l) of LV-Gag once at day l or twice (day 1 and day 2).
  • the single inoculation killed 9/10 mice, whereas only 4/10 died after receiving a double dose of virus.
  • Control mice were inoculated with the backbone pCV-0 plasmid DNA alone or combined with K2 and boosted with alum alon.
  • Two weeks after last immunization were challenged with LV-Tat and observed for disease onset and death. The results of this experiment are promising since 3/10 died in the group immunized with pCV-Tat ⁇ K2 at day 22 after challenge, as compared to 4/10 which died in the group inoculated with pCV-0 ⁇ K2.
  • HSV-1 recombinant viruses which are more pathogenic that HSV-1 ⁇ UL41
  • HSV-1 deleted in a different non-essential viral locus eg the Us5 locus corresponding to glycoprotein JI (gJl)
  • gJl glycoprotein JI
  • These virus expresses the lacZ reporter gene under the control of HCMV-imediate early gene promoter ( Figure 12) and is currently beign tested for pathogenicity in mice in comparison to HSN-l ⁇ UL41-lacZ and wild- type HSN-1.
  • mice syngeneic (C57) HIN-1 MuLVinfected spleen cells they produce HIV-1 pro viral D ⁇ A, able to infect mice spleen cells.
  • the successfully vaccinated mice cleared HIV-1 infected spleen cells 10-14 days after the challenge.
  • the second one is based on infravenous inoculation of vaccinated Balb-c mice with replication competent vaccinia virus encoding HIV-1 genes. The end-point is the inhibition of viral growth.
  • HSV can infect and establish a latency in mice, but also kill them if given at the appropriate dose (Kuklin et al, 1998) and its genome is well known, so it is quite easy to handle with it.
  • the results obtained with the recombinant HIV/HSV-1 with deletion in UL41 indicate that such viruses are able to infect and kill mice, if administrated at the appropriate dose and through the right route of inoculation.
  • the data obtained from the chllenge experiment also suggest that not all the recombinant viruses have the same behavior: if they encode for a structural protein (like Env or Gag) the titer needed for mice killing seem to be lower than the one necessary with the virus expressing a regulatory protein as Tat, that is known to have a plethora of effect on the immune system and on the cell cycle and to be implied in the confrol of viral gene expression, replication and pathogenicity.
  • LV-HIN viruses have shown to kill the animals the dose to be use is very high to respect to LV wild-type virus suggesting that the mutation in vhs gene (UL41) of HSV-1 or HSV-2 viruses attenuates the pathogenicity of the wild-type viruses.
  • New viruses have been constructed with deletion in sequences that should do not reduces replication and susceptibility in mice.
  • These LV-HSV-1 or G-HSV2 new mutants are deleted in a small portion of glycoprotein gGl that is a non-essential for viral replication. Mutations in Us4 (gG) or Us5 (gJ) should do not interfere with HSV replication in vivo allowing the use of low viral doses with higher efficiency.
  • HSV-1 recombinant vectors expressing HIV-1 genes in the UL41 locus HSV-1 recombinant vectors expressing HIV-1 genes in the UL41 locus.
  • mice inoculated (A) by intravaginal or (B) intraperitoneal route with three different doses of LV- Tat.
  • LV-Tat virus given infravaginally at the dose of 1.4 x 10 8 , is able to kill 5/7 mice. None of the tested doses is able to kill the mice if given infraperitoneally.
  • mice inoculated by the intravaginal route with the same dose of LV-Gag given once or twice. In addition, mice were inoculated with the same dose by the intraperitoneal rout.
  • FIG.12
  • Example 5 Expression of Human Immunodeficiency Virus type 1 tat from a replication-deficient Herpes simplex type 1 vector induces antigen-specific T cell responses
  • the he ⁇ es simplex virus (HSV) vectors show several advantages for prophylaxis against viral infections. They have been shown: i) to elicit strong and durable immune responses by various routes of inoculation [39,44]; ii) the viral D ⁇ A persists inside the host's cell nucleus as an episomal element, thus eliminating the safety concerns deriving from the random integration of the viral genome into the host's D ⁇ A; iii) they carry the tk gene, encoding the viral thymidine kynase, that, in case of undesired effects, can be used, in combination with specific antiviral drugs, to kill the virus-harbouring cells.
  • HSV vectors require the development of mutated viruses that are genetically stable, uncapable of replicating in the C ⁇ S and of spreading in immuno-compromised individuals, not transmissible from immunized individual by contacts and, at the same time, capable of inducing protective immunity against the disease.
  • replication-defective he ⁇ es simplex viruses characterized by the simultaneous deletion of multiple viral functions, including the immediate-early proteins ICP4, ICP27, ICP22 and the structural protein VHS (viral host shutoff) have been developed [45,46].
  • HSV mutants show a reduced cytotoxicity , due to their inability to replicate and to spread in the host, but maintain the capability to infect a wide range of tissues and host species, h addition, these recombinant replication-defective vectors sustain high expression of the exogenous genes under homologous or heterologous promoters (HSV-1 or HCMV, respectively) [47,48], and because of their large genome can be arranged to simultaneously express multiple antigens [49]. Moreover, recent studies indicate that the pre-existing immunity against HSV infection does not compromise its efficacy as a vaccine vector [50,51].
  • Plasmid pCN-tat expressing the HIN-1 t ⁇ t cD ⁇ A (HTLN-IIIB isolate, subtype B) has been previously described [52,53]. Plasmid D ⁇ A was purified from Escherichia coli by using Qiagen endotoxin free Maxi Kit (Qiagen, Hilden, Germany).
  • Plasmid pB410-tat was constructed by introduction of the HIN-1 tat cD ⁇ A (350 pb) from pCV- tat into the UL41 locus of plasmid HSN-1 pB41 that has been described elsewhere [47].
  • the tat cD ⁇ A under the transcriptional control of the HSN immediate-early ICPO promoter was inserted into EcoRI/Xbal sites of pB41 plasmid between the two UL41 HSN fragments (HSN genomic positions 90.145-91.631 and 92.230-93.858) [54].
  • the TOZGFP is a replication-defective HSN-1 viral vector having a low cytotoxicity due to the deletion of three immediate early genes (ICP4, ICP27, which are essential for viral replication and ICP22 which is not) and contains the gfp gene in the ICP22 locus and also the LacZ gene in the UL41 locus as marker genes.
  • Plasmid pB410-tat was constructed to genetically recombine with the genome of the TOZGFP viral vector using the previously described Pac-facilitated lacZ substitution method [54].
  • the generation of recombinant viruses was carried out using the standard calcium phosphate transfection procedure with 5 ⁇ g of TOZGFP viral D ⁇ A and 1 ⁇ g of linear plasmid pB410-tat. Transfection and isolation of the recombinant viral progeny was performed in 7b cells as previously described [45,55].
  • the recombinant virus TO-tat containing the tat cD ⁇ A was first identified by isolation of a clear plaque phenotype after X-gal staining.
  • the TO-tat virus was purified by three rounds of limiting dilution technique and the presence of the transgene was confirmed by Southern blot analysis.
  • Viral stocks of the TO-tat and of the control vector TO-GFP were prepared and titrated using Vero-ICP4 and ICP27 stabely- transfected 7b cells [55].
  • the Vero-derived cell line termed 7b, expresses the HSV-1 immediate early genes ICP4 and ICP27 required for virus replication [45,55]. .
  • the monkey kidney fibroblast or (Vero) the 7b cells, the P815 murine (H-2 d ) mastocytoma cell line, the baby hamster kidney cells (BHK) and the fibroblasts balb/c cells were all cultured in DMEM (Euroclone, Grand Island, NY) supplemented with 10% FBS (Euroclone), 2mM L-glutamine, 100 Dg/ml penicillin and 100 TJ/ml streptomycin. 7b cells were subjected montly to two-weeks long selection with 1 mg/ml G418 (Sigma).
  • Human HeLa 3T1 cells were grown in DMEM and 10% FBS; these cells contain an integrated copy of plasmid HIV-LTR-CAT where expression of the choramphenicol acetyl fransferase (CAT) reporter gene is achievable only in the presence of Tat, indispensable for fransactivating the HIN-LTR promoter driving CAT [56].
  • Splenocytes from immunized and control mice were cultivated in RPMI 1640 (Euroclone) supplemented with 10% Hyclone (Euroclone), 50 ⁇ M ⁇ -mercaptoethanol, and 10 mM HEPES.
  • Tat protein expression from the TO-tat vector was analyzed in murine fibroblast cell line BALB/c cells (lxlO 6 cells) infected with TO-tat virus at multiplicity of infection 1 (m.o.i.l).
  • Cell extracts corresponding to 10 ⁇ g of total proteins, were loaded on 12% SDS-polyacrylamide gel and analyzed by Western blot using a rabbit anti-Tat polyclonal serum (Intracel) at 1:1000 dilution and a mouse anti-rabbit HRP-conjugated secondary antibody (Sigma) at 1 :4000 dilution.
  • Tat antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 1 with TO-GFP recombinant vector.
  • Tat protein was determined by Cat assay as follows: HeLa 3T1 cells (lxlO 6 ) were infected in suspension with TO-tat and control vector TO-GFP, at different m.o.i. (from 0.01 to 1) for 1 hour at 37°C under mild shaking. After infection, cells were washed twice with complete medium to eliminate the virus particles that did not infect the cells, plated onto 6 well-plates, and cultured at 37°C for 24 and 48 hours. After incubation, cells were disrupted by sonication, supematants were collected and cellular debris removed by cenfrifugation. CAT expression was measured after normalization to protein content (100 ⁇ g aliquots) as previously described [57].
  • Recombinant HIN Tat from the HTLV-IIIB isolate (subtype B) was expressed in Escherichia coli, purified to homogeneity by heparin-affinity chromatography and high-performance liquid chromatography and stored lyophilized at -80°C as described [58].
  • Purified Tat protein had full biological activity in several assays [14,59,60]. As Tat is sticky, easily oxidable and photo- and thermo-sensitive, it was resuspended at 2 ⁇ g/ml in degassed PBS containing 0,1% BSA immediately before use and handled on ice and in the dark, with degassed buffer pre-flushed plasticware.
  • HSV-1 stocks were prepared by infecting 4x10 s 7b complementing cell lines with 0,05 MOI of TO-tat and TO-GFP viruses in suspension in 15 ml of medium for 1 hour at 37°C under mild agitation. When a 100% cytopathic effect was evident, cells were collected and centrifuged at 2000 ⁇ m for 15 minutes. The supematants were spun at 20.000 rpm in JA20 rotor (Beckman) for 30 minutes to collect the vims. The cellular pellets were resuspended in 2 ml of medium, subjected to three cycles of freeze-thawing (-80°C/37°C) and a single burst of sonication, to release the viral particles.
  • the virus was further purified by density gradient centrifugation (Opti Prep; Life Technologies, Inc.) and resuspended in PBS-A IX. Viral stocks were titered as previously described (55) and stored at -80°C. Titles averaged between 2xl0 8 to 2xl0 9 plaque forming unit pfu/ml
  • mice Animals were handled according use was according to national guidelines and institutional policy. Six weeks old BALB/c (H-2 d ) female mice were purchased from Harlan Italy and immunized after one week according to the protocols described below.
  • mice were immunized with 4xl0 4 pfu or 4xl0 6 pfu of TO-tat purified vims by subcute (s.c.)-injection on the left flank.
  • Control animals were injected with PBS.
  • the animals were boosted s.c. at weeks 2, 4, and 9 after priming immunization.
  • mice were immunized with 4xl0 6 PFU of TO-tat vims or PBS, s.c. on the left flank or intranasally (i.n.) The animals were boosted s.c. and i.n. respectively at weeks 2, 4, and 9 after priming.
  • mice were immunized s.c. with 4xl0 6 PFU of TO-tat vims or PBS. Animals were boosted s.c. at weeks 2, 4, and 9, or at weeks 4 and 8 after priming.
  • Fourth immunization protocol mice were primed with 4xl0 6 PFU of TO-tat vims by s.c. route, or with recombinant Tat protein (2 ⁇ g/mouse) by the intradermal route (i.d.). Confrol animals were injected with PBS. All mice were boosted s.c. at weeks 2and 9 after priming with 4xl0 6 PFU of TO-tat vims.
  • mice per group were sacrificed at day 14 after each boost immunization to collect spleens, vaginal fluids and blood samples for analysis of the immune responses of individual mice.
  • Anti-Tat IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA).
  • concentration of the recombinant protein used for coating was 1 ⁇ g/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8), and 100 ⁇ l/well were added to 96-well immunoplates (Nunc- Immunoplate F96 Polyso ⁇ , Nunc, Naperville, IL).
  • the plates were sealed and incubated in the dark for 18 to 20 hours at 4°C. Prior to use, the plates were extensively washed with 0.05% Tween 20 in PBS-A IX and blocked for 90' at 37°C with 3% bovine serum albumin (BSA) in PBS-A IX.
  • BSA bovine serum albumin
  • mice spleens were disrupted with 2ml syringe plungers using 70 ⁇ m pores cell strainers (Falcon), resuspended in PBS IX with 2 mM EDTA and, after 15' cenfrifugation at 1500 ⁇ m, treated with red blood cell lysis buffer (100 mM NH4C1, 10 mM KHCO3, 10 ⁇ M EDTA) for 4 minutes at room temperature and finally washed with RPMI 1640 medium (Euroclone) containing 3% of heat-inactivated FBS.
  • red blood cell lysis buffer 100 mM NH4C1, 10 mM KHCO3, 10 ⁇ M EDTA
  • Splenocytes (2xl0 5 cells/well) were cultured in 96 well plates in the presence of 1 ⁇ g/ml and 5 ⁇ g/ml of recombinant Tat protein, 10 ⁇ g/ml of Con A (ICN) or culture medium alone as positive and negative controls respectively.
  • ICN Con A
  • bromodeoxyurindine (BrdU) was added (10 ⁇ M/final concenfration) to the plates.
  • BrdU inco ⁇ oration was determined after ON incubation with BrdU by using a cell proliferation ELISA system (Amersham Pharmacia Biotech) according to the manufactures instructions.
  • mice splenocytes were co-cultivated at 1.5:1 ratio with naive syngeneic stimulator splenocytes, previously irradiated at 30 Gy, in the presence of 1 ⁇ g/ml of purified recombinant Tat protein.
  • Recombinant IL-2 (10 U/ml) was added to the cells after 3 days of culture.
  • 5I Chromium release assays were performed at day 6 of culture using P815 target cells, preincubated overnight with 2 ⁇ g/ml of Tat, as previously described [61], with additional 10 ⁇ g of protein during the 51 Cr (100 ⁇ Ci/target) labeling step. After 4 hours incubation of effector and target cell at 37°C, supematants were harvested and the 51 Cr released by the lysed target cells was quantified using a ⁇ -counter. Specific percent cell lyses was calculated according to the following formula:
  • the cytokine profile was determined in culture supematants of mice splenocytes (2,5x10 6 /l ml in 48 well plate) cultured with 1 ⁇ g/ml of recombinant Tat protein, 10U hIL-2 from day 3. At day 3 and 6 of culture standard sandwich ELISA tests were performed, using antibodies and recombinant standard proteins purchased from ENDOGEN. The concenfration of the anti-IFN ⁇ antibody used for coating was 1 ⁇ g/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8), and for anti-IL-4 2 ⁇ g/ml diluted in PBS IX.
  • TO-tat replication defective HSV-1 virus was modified in order to express the HIV-1 Tat protein under the confrol of the HSV-1 immediate early (ICPO) promoter (TO-tat) (Fig.13).
  • This recombinant vims named TO-tat was obtained by homologous recombination of the pB410-tat plasmid with the HSV-1 TOZ-GFP triple mutant vims into UL41 HSV-1 locus.
  • Tat protein was assessed by infecting Balb/c fibroblasts with TO-tat replication-defective HSV-1 recombinant vector and Tat expression analysed by Western blot after 24, 48 and 72 hours post- infection. Controls were represented by cells infected with the TO-GFP vector or non-infected cells. As shown in the Figure 14A the TO-tat vector expressed Tat at high levels. Tat expression was detected already 24 hours after infection and increased up to 72 hours post infection. Similar patterns of Tat expression were detected in Vero, BHK, HeLa and P815 cells (data not shown).
  • Balb/c fibroblast were grown for 4, 6, 8, 12 and 18 hours with cell-free culture supematants (collected at 48 hours post infection) of TO-tat infected cells. Internalization of Tat by Balb/c cells was then analysed at each time-point on cell lysates by Western blot. Negative control was represented by Balb/c cells cultured for 8 hours with supernatan derived from TO-GFP infected cells. As shown in figure 14B TO-tat infected cells release Tat in the extracellular medium, and this extracellular released protein is efficiently taken up by uninfected cells.
  • Tat is promptly detected intracellularly in uninfected Balb/c cells after 4 hours incubation with Tat-containing supematants and its uptake increases up to 8 hours incubation. After 12 and 18 hours Tat become undetectable likely because cells, which have internalized the protein, had already processed by that time.
  • HeLa3Tl cells containing an integrated copy of the CAT reporter gene under the transcriptional control of the HIV-LTR promoter, and in which CAT expression occurs only in the presence of bioactive Tat, were infected with different m.o.i. (0.01-1) of TO-tat or TO-GFP confrol vector. CAT expression was measured 24 and 48 hours post-infection.
  • Table 1 Analysis of the biological activity of Tat expressed by TO-tat a
  • mice were immunized with 4x10 or with 4x10 pfu by the s.c. route and boosted at weeks 2, 4 and 9 after priming.
  • the analysis of the immune responses elicited in animals have demonstrated that only the group of mice immunized with the higher dose of TO- tat recombinant vims was able to mount a significant response against Tat (data not shown). From these preliminary studies the dose of 4xl0 6 pfu recombinant vims was chosen for the subsequent experiments.
  • mice were vaccinated with 4xl0 6 pfu of TO-tat by s.c. or i.n. inoculation. Mice were boosted with the same dose of TO-tat s.c. or i.n. at weeks 2, 4 and 9 after priming (Fig. 15A).
  • FIG. 15A The results of this experiment, shown in figures 15B and 15C, indicate that only mice vaccinated with TO-tat by the s.c. route developed a specific immune response to Tat. Antigen-specific CTL responses and TNF ⁇ production were detected in mice vaccinated s.c.
  • mice vaccinated i.n. with TO-tat did not develop any specific anti-tat response in a fashion similar to control mice injected with PBS.
  • IL-4 production was barely detectable or undetectable in all groups (Fig. 15C).
  • a possible explanation for the different responses after s.c. and i.n. immunization may be that immunization by i.n. route might require a higher dose of recombinant virus in order to induce a specific response against Tat or that the immunization by i.n. route should be implemented with an adequate mucosal adjuvant to- achieve the desired effect.
  • mice were inoculated with purified Tat protein by the intradermal route and boosted twice with 4xl0 6 TO-tat viral particles by s.c. route (heterologous regimen) (Fig. 17A).
  • mice were immunized three times with 4x10 6 pfu of TO-tat vims by s.c. route (homologous regimen) (Fig. 17A).
  • the results indicate that one effective CTL response was elicited only by the homologous (vaccination regimen) and not by the heterologous immunization regimen (Fig. 17B).
  • mice vaccinated with the homologous regimen (Fig.l7C).
  • the production of IL-4 was either barely detectable or undetectable in all groups of mice (Fig. 17C).
  • the differences in the immunological response between homologous and heterologous regimen may depend on the fact that (no adjuvant was used with the protein inoculation), as expected, the viral TO-tat vector has proven to be much more efficient in the priming of a Thl -like immune response, characterized by anti-Tat CTL activity and INF ⁇ induction.
  • Tat-specific T cell proliferation was evaluated by BrdU inco ⁇ oration in mice splenocytes cultured with recombinant Tat. No significant T cell proliferation was detected in TO-Tat immunized animals at any viral dose, administration route or vaccination protocol (data not shown), suggesting that HSV-1 derived vaccines are weak inducers of T-dependent antibody responses.
  • HSV recombinants as vaccination vectors is based on the fact that the viral Vhs protein (viral host shutoff) encoded by UL41 gene has been shown to block dendritic cell (DC) maturation and thus inhibit the immune response against the vector-delivered transgene [70].
  • DC dendritic cell
  • the purified Tat protein prime/TO-tat viras boost heterologous vaccination regimen was not so efficient in inducing the Tat-specific cellular immunity, resulting in generally low, yet still detectable CTL activity and INF- ⁇ production in the restimulated lymphocyte cultures after the complete time course of the immunizations. Furthermore, low to intermediate levels of anti-Tat antibodies were detected in the sera of immunized mice. It is likely that, in order to improve the overall, i.e. both cellular and humoral, efficacy of the heterologous vaccination model, we will need to associate an adjuvant agent to the recombinant protein during the priming inoculations.
  • HSN-1 vectors Another protein or recombinant viras boost might be required to obtain an efficient and complete immune response against a weakly immunogenic HIN-1 Tat protein.
  • the appealing properties of replication incompetent HSN-1 -based vectors inducing strong CTL response, both in murine and in simian model, against foreign genes delivered by viral particles have made them very promising candidates for potential anti-HIV-l and also other viral or intracellular bacterial pathogens vaccine development. Further studies will have to be performed in order to investigate the feasibility of HSN-1 vectors application for human use, too.
  • FIG. 13 Schematic representation of pBlueScript plasmid containing UL41-ICP0-tat cassette (A) and of the TO-GFP and TO-tat HSN-1 replication defective vector (B).
  • Expression of GFP gene is driven by HCMV IE promoter.
  • the black squares symbolize the IE genes (ICP4, 27, 22) and other genes that are deleted in the HSV backbone.
  • the white squares symbolize the terminal and internal repeats of the HSV genome delimiting the unique regions (U L : unique long; Us: unique short)
  • Fig. 14 Western blot analysis of Tat protein expressed by the TO-tat HSV-1 vector.
  • A Balb/c cells infected with 1 m.o.i of TO-tat and analyzed at 24 (lane 1), 48 (lane 2) and 72 (lane 3) hours post-infection. Control cells were infected with 1 m.o.i. of the TO-GFP vector (lane 4). Recombinant Tat protein was loaded as the positive confrol at 20 ng (lane 5), 50 ng (lane 6).
  • B analysis of Tat protein uptake in Balb/c cells cultured with media obtained from TO-tat infected cells.
  • FIG. 15 Analysis of anti-Tat immune responses elicited by vaccination with 4.x 10 6 pfu of TO-Tat HSV-1 replication defective vims subcutaneously (s.c.) or infranasally (i.n.).
  • B Tat-specific CTL response analyzed by 51 Chromium release assays
  • C INF ⁇ and IL-4 production in cell-free culture supematants of mice splenocytes after 6 days in vitro culture.
  • B and C the data shown in charts mean results conespond to the mean ( ⁇ SD) of individual mice.
  • Tat HSV-1 replication defective viras in different immunization schedules Tat HSV-1 replication defective viras in different immunization schedules.
  • A schematic representation of the homologous prime/boost immunization schedule. Mice were immunized with TO-tat and boosted at weeks 2,4 and 9 (schedule 1) or at weeks 4 and 8 after the first inoculation (schedule 2).
  • B Tat-specific CTL responses measured by 51 Chromium release assays
  • C INF ⁇ and IL-4 production in cell-free culture supematants obtained from mice splenocytes after 6 days in vitro culture with recombinant Tat at 1 ⁇ g/ml.
  • B and C results are represented as the mean ( ⁇ SD) of individual mice.
  • Fig. 17 Analysis of anti-Tat immune responses after homologous or heterologous prime/boost vaccination regimens with 4.x 10 6 pfu of TO-Tat HSV-1 replication defective vector.
  • Fig. 18 Tat-specific IgG antibodies in the sera of Balb/c mice immunized with TO-tat replication- defective HSV-1 vector.
  • Sera were obtained from mice primed with TO-tat and boosted with the same viras (4xl0 6 viras particles) s.c, or from those that have been Tat protein-primed and boosted with TO-tat.
  • Confrol mice were injected with PBSX1.
  • ELISA assays were performed as described in the material and methos section andthe absorbabce values of four representative mice/group are shown References for Example 5
  • HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix- associated heparan sulfate proteoglycans through its basic region. Aids 1997, 11(12), 1421-1431.
  • the he ⁇ es simplex type-1 viras (HSN-1) vectors show several advantages for prophylaxis against viral infections. They have been shown: i) to elicit strong and durable immune responses by various routes of inoculation; ii) the viral D ⁇ A persists inside the host's cell nucleus as an episomal element, thus eliminating the safety concerns deriving from the random integration of the viral genome into the host's D ⁇ A; iii) they carry the tk gene, encoding the viral thymidine kinase, that, in case of undesired effects, can be used, in combination with specific antiviral drags, to kill the virus-harbouring cells.
  • viruses employed in the following invention deserve specific description.
  • ICP4 ICP4 " , ICP27 " , and ICP22 " vims having an ICP4-immediate early promoter-tk gene expression cassette at the UL24 locus known in art (Marconi, P., Krisky, D., Oligino, T., Poliani, P.L., Ramakrishnan, R., Goins, W.F., Fink, D.J., 8c Glorioso, J.C. (1996). Replication- defective he ⁇ es simplex viras vectors for gene transfer in vivo. Proc Natl Acad Sci U S A, 93(21), 11319-11320.).
  • the HSN-1 vectors have been modified by insertion of heterologous expression cassettes encoding for HIN-1 proteins at the UL41 or ICP22 locus of HSN-1 replication defective vector.
  • a D ⁇ A fragment comprising an HSN ICPO-immediate early promoter-lacZ expression cassette flanked by Pad restriction endonuclease recognition sites and sequences homologous to UL41 for HSN-1 was co-trasfected in 7b cells with HSN-1 mutant. Recombinants were screened for lacZ expression, and the correct insertion was confirmed with Southern blot analysis.
  • Plasmid p239SpSp5', expressing the SIV-1 gag cD ⁇ A has been previously described (Kestler HW III, Kodama T, Ringler D, Marthas M, Pedersen ⁇ , Ratner A, Regier D, Sehgal T, Daniel M, King ⁇ , Desrosiers RC, (1990). hiduction of AIDS by molecularly cloned vims. Science, 248: 1109-1112.). Plasmid D ⁇ A was purified from Escherichia coli by using Qiagen endotoxin free Maxi Kit (Qiagen, Hilden, Germany).
  • Plasmid pB410-gag was constructed by introduction of the 1.9 kbp SIV-1 gag cD ⁇ A (SIV mac 239 proviras genome) after subcloning the sequence respectively in pSP72 and pcD ⁇ A3-Gag expression cassette into the UL41 locus of HSV-1.
  • the cDNA under the transcriptional confrol of the human cytomegalovirus promoter (NruI-EcoRI) was inserted in a S ⁇ I/EcoR/-opened pBBSK-plasmid between the two UL41 fragments (map positions 93,858-92,230 and 91,631- 90,145) 100 bp downstream of the HSN-1 immediate-early ICPO promoter.
  • TO-GFP is a non-replicative HSV-1 viral vector that has low toxicity due to the deletion in three immediate early genes (ICP4, ICP27, which are essential for viral replication, and ICP22) with cD ⁇ A encoding GFP inserted into the ICP22 locus and an insertion of LacZ in the UL41 locus.
  • the recombination was carried out using standard calcium phosphate fransfection of 5 ⁇ g of viral D ⁇ A and 1 ⁇ g of linear recombination plasmid pB410Hgag.
  • Transfection and isolation of the recombinant vims was performed in 7b Vero cells (African green monkey kidney cells CCL81: ATCC, Rockville, MD) capable of providing the essential ICP4 and ICP27 HSN-1 gene products.
  • the recombinant vims containing the gag cD ⁇ A was identified by isolation of a clear plaque phenotype after X-gal staining.
  • the viras was purified by three rounds of limiting dilution and the presence of the transgene was verified by Southern blot analysis.
  • PB5 is a pBSSK plasmid containing the Usl HSV- 1 sequences encoding the ICP22 immediate early protein, as previously described (ref: Krisky DM, et al.
  • THZ-1 is a recombinant vims deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMV promoter, in the ICP22 locus.
  • THZ-1 viral D ⁇ A and the recombinant plasmid containing the gmcsf gene have been co-transfected into 7b complementing cell line.
  • the TH-gmcsf viras was purified by three rounds of limiting dilution and the presence of the transgene was verified by Southern blot analysis. Western blot analysis and ELISA of the recombinant vims-infected cell lines was performed in order to assess the transgene expression.
  • HSV-1 recombinant vectors expressing SIV-1 Gag and murine GM-CSF Construction of HSV-1 recombinant vectors expressing SIV-1 Gag and murine GM-CSF
  • the vector TOHgag Hgmcsf containing SIV-1 gag in UL41 HSV-1 locus and gmcsf in Usl HSV-1 locus was created by genetically crossing the TOHgag and THgmcsf vectors. 7b cells plated on 60 mm petri dishes were infected with 3 m.o.i. of TOHgag and THgmcsf viruses and harvested 18 hours post infection. The mixture of viruses derived from the co-infection was titrated, and the viral vector containing both SIV-1 gag and murine gmcsf genes was isolated by Southern blot screening.
  • SIV-1 Gag protein expression was analysed in Vero cells (1x10° cells/well in 6 well plates) infected with 1 m.o.i. TOHgag vims. At 12, 24, 48 and 72 hours after infection cell exfracts corresponding to 10 ⁇ g of total protein, were run onto 12% SDS-polyacrilamide gel and transferred by elecfroblotting to a polyvinylidene difluoride membrane (Millipore, Bedford, MA).
  • the blots were stained with a mouse monoclonal anti-SIN-p27specific antibody (ENA 643, Centralised facility for AIDS Reagents), diluted 1:2000 and a goat anti mouse IgG HRP- conjugate secondary antibody ( ⁇ A931, Amersham) diluted 1:2500. Immunocomplexes were detected by ECL western blot detection kit (Amersham, Pharmacia Biotech). Controls was represented by Vero cells infected with 1 m.o.i. of TO-GFP recombinant vector and collected at 48 hours after infection.
  • HSV-1 stocks were prepared by infecting 4xl0 8 7b complementing cells with 0,05 M.O.I. of TOHgag and TO-GFP viruses in suspension in 15 ml of medium for 1 hour at 37°C under mild agitation. When a 100% cytopathic effect was evident, cells were collected and centrifuged at 2000 rpr ⁇ for 15 minutes. The supematants were spun at 20.000 ⁇ m in JA20 rotor (Beckman) for 30 minutes to collect the viras.
  • the cellular pellets were resuspended in 2 ml of medium, subjected to three cycles of freeze-thawing (-80°C/37°C) and a single burst of sonication, to release the viral particles.
  • the vims was further purified by density gradient centrifugation (Opti Prep; Life Technologies, Inc.) and resuspended in PBS-A IX. Viral stocks were titered and stored at -80°C Titles averaged between 2x10 to 2x10 plaque forming unit (pfu)/ml.
  • mice Animals were handled according to national guidelines and institutional policy. Six weeks old BALB/c (H-2 d ) female mice were purchased from Harlan Italy and immunized when they were 7 weeks old, according to the protocols described below.
  • mice Female 7-weeks old BALB/c mice (Harlan Italy) were primed by subcutaneous (s.c.) or intradermal (i.d.) route with TOHgag or TO-GFP (4xl0 6 viras particles/100 ⁇ l of PBS-A lx).
  • mice were boosted s.c or i.d. after 15 days with the same dose of immunogen.
  • the recombinant viruses were administered in 100 ⁇ l for the s.c. (one site) and i.d. (50 ⁇ l / site) routes. Seven mice per group were sacrificed on day 14 and after the boost on day 28 to collect spleens, vaginal fluids and blood samples for analysis of the immune responses of individual mice. Serology
  • Anti-Gag IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA) as previously described (O'Hagan D. et al. J. of Virology 2001, vol.75 (19): 9037-9043) .
  • the concenfration of the recombinant protein used for coating was 5 ⁇ g/ml diluted in PBS- A IX, and 50 ⁇ l/well were added to 96-well immunoplates (Nunc-Immunoplate Maxiso ⁇ , Nunc, Naperville, IL) and incubated over night, in the dark at 4°C Prior to use, the plates were extensively washed with 0.1% Tween 20 in PBS-A IX and blocked for 60' at 37°C with 1% normal serum goat in 0.1% Tween 20 and PBS-A IX.
  • Sera were 1:100 diluted in blocking buffer and each sample was run in triplicate wells (50 ⁇ l/well). After incubation at 37°C for 120', the plates were washed and immunocomplexes were detected with 50 ⁇ l/well of anti-mouse IgG HRP conjugate (Amersham NA931) diluted 1:20.000 in blocking buffer. After incubation at 37°C for 60 minutes, the wells were washed, and incubated at room temperature for 30 minutes with 50 ⁇ l/well of TMB (Sigma) as HRP substrate. The reaction was blocked with 50 ⁇ l of HCL IN per well. The absorbance was measured at 450 nm with an automated plate reader (ELX-800, Bio-Tek Instruments, Winooski, UT).
  • the positive control was represented by a recombinant Gag (SIN p27-GST fusion protein - NIBSC EVA 643) and the negative control was represented by the sera from mice injected with T0GFP vector and PBS-A lx. Absorbance values higher than the control group (PBS-A lx injected) mean + 3SD values were considered positive.
  • mice spleens were disrupted with 2ml syringe plungers using 70 ⁇ m pores cell strainers (Falcon), resuspended in PBS-A IX with 2 mM EDTA and, after 15' cenfrifugation at 1500 rpm, treated with red blood cell lysis buffer (100 mM NH4C1, 10 mM KHCO3, 10 ⁇ M EDTA) for 4 minutes at room temperature and finally washed with RPMI 1640 medium (Euroclone) containing 3% of heat-inactivated fetal bovine serum.
  • red blood cell lysis buffer 100 mM NH4C1, 10 mM KHCO3, 10 ⁇ M EDTA
  • Splenocytes (2x10 5 cells/well) were cultured in 96 well plates in the presence of 1 and 5 ⁇ g/ml of each gag peptide, divided in two pools, or with, 10 ⁇ g/ml of Con A (ICN) or culture medium alone as positive and negative controls respectively.
  • ICN Con A
  • bromodeoxyuridine (BrdU) was added (10 ⁇ M/final concenfration) to the plates.
  • BrdU inco ⁇ oration was determined after an over night incubation by using a cell proliferation ELISA system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
  • mice splenocytes were co-cultivated at 1.5:1 ratio with naive syngeneic stimulator splenocytes, previously irradiated at 30 Gy and pre-incubated for one hour with in the presence of 10 ⁇ g/ml of the pool 1 or the pool 2 of Gag peptides.
  • Recombinant IL-2 (10 U/ml) was added to the cells after 3 days of culture.
  • 51 Chromium release assays were performed at day 6 of culture using P815 target cells, preincubated with 10 ⁇ g/ml of each Gag peptide and 100 ⁇ Ci of Na 2 CrO 3 (NEN).
  • the cytokine profile was determined in culture supematants of mice splenocytes (2,5x10 6 /l ml in 48 well plate) cultured with 10 ⁇ g/ml of each gag peptide, 10U hIL-2 from day 3. At day 3 and 6 of culture standard sandwich ELISA tests were performed, using antibodies and recombinant standard proteins purchased from ENDOGEN.
  • the concentration of the anti-IFN ⁇ antibody used for coating was 1 ⁇ g/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8).
  • the anti- IL-4 antibody was diluted in PBS-A IX at 2 ⁇ g/ml.
  • a replication defective HSV-1 viras was modified in order to express the SIV-1 Gag protein under the control of HCMV immediate early promoter, 100 bp downsfream of the HSV immediate-early ICPO promoter (Fig.l9A).
  • This recombinant viras named TOHgag was obtained by homologous recombination of the pB410-gag plasmid with the HSV-1 TOZ-GFP triple mutant viras into UL41 HSV-1 locus.
  • the presence of the gag gene in the HSV-1 genome was confirmed by Southern blot analysis.
  • telomere expressing murine GM-CSF was constracted by co-transfecting a plasmid (PB5-gmcsf), which contains gmcsf gene under HCMV promoter flanked by ICP22 HSV-1 sequences, with THZ1 recombinant backbone viras into 7b complementing cell line.
  • THZ-1 is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the lacZ reporter gene, under the control of HCMV promoter, in the ICP22 locus (Fig.l9B).
  • the vector TOHgag/Hgmcsf containing the gag gene in UL41 HSV locus and gmcsf in Usl HSV locus was created by genetically crossing the above vectors (Fig.l9C).
  • Gag protein was assessed by infecting Vero fibroblasts with T0H:gag replication- defective HSV-1 recombinant vector and Gag expression analysed by Western blot after 12, 24, 48 and 72 hours post-infection. Controls were represented by cells infected with the TO-GFP vector or non-infected cells. As shown in the Figure 20 the T0H:gag vector expressed Gag at high levels. The protein expression was revealed by a mouse monoclonal anti-SIV-p27specific antibody (EVA 643, Centralised facility for AIDS Reagents), diluted 1:2000 and a goat anti mouse IgG HRP-conjugate secondary antibody (NA931, Amersham) diluted 1:2500. Immunocomplexes were detected by ECL
  • mice were immunized with 4x10 6 pfu by the route and boosted also s.c. or i.d. two weeks after priming.
  • the analysis of the immune responses elicited in animals has demonstrated that only the group of mice immunized i.d. after the priming was able to mount a significant response against Gag (Fig. 22).
  • the group of mice immunized by s.c. route after the s.c. boost has demonstrated a significant and much stronger immune response in comparison with the i.d. prime/boost vaccination regimen (Fig. 23A and 5B).
  • mice vaccinated s.c. or i.d. with TOHgag High levels of INF- ⁇ were detected in splenocytes of mice vaccinated s.c. or i.d. with TOHgag, while mice vaccinated s.c. or i.d. with TO-GFP did not develop any specific anti-Gag response in a fashion similar to control mice injected with PBS-A lx (Fig.24A and 25A). IL-4 production was low but detectable in the mice immunized with both TOHgag groups (Fig. 24B and 25B).
  • Gag-specific T cell proliferation was evaluated by BrdU inco ⁇ oration in mice splenocytes cultured with Gag peptides. No significant T cell proliferation was detected in TOHgag immunized animals at any viral administration route suggesting that HSV-1 derived vaccines are weak inducers of T helper-mediated responses, and among them the antigen-specific antibody production.
  • T0H:gag replication-defective HSV-1 recombinant vector was able to elicit a Gag-specific immune response in immunized mice, although the breadth of the response was different depending on the site of inoculation.
  • FIG. 19 Schematic representation of recombinant TOHgag viras.
  • A schematic representation of HSN-1 genome, containing SIN-1 gag cD ⁇ A under the control of HCMV fused to 100 bp of HSV-1 ICPO promoter into HSV-1 UL41 locus. Expression of GFP gene is driven by HCMV IE promoter.
  • the red squares symbolize the IE genes (ICP4, 27, 22) and the green squares other genes that are deleted in the HSV-1 backbone.
  • the white squares symbolize the terminal and internal repeats of the HSV-1 genome delimiting the unique regions (U L : unique long; Us.” unique short)
  • Fig. 20 Western blot analysis of SIV-1 Gag protein expressed by the TOHgag HSV-1 vector.
  • Vero cells were infected with 1 m.o.i of TOHgag and TO-GFP (as negative confrol).
  • Primary antibody anti-SIV-1 p27 lmicrogr/ml (EVA 643, Centralised facility for AIDS Reagents); secondary antibody: antimouse IgG HRP-conjugate ( ⁇ A931V, Amersham) 1:2500.
  • Lanes 1-4 T0GAG infected Vero cells, 72, 48, 24 and 12h post infection, respectively; lane 5: MW marker; lane 6: T0GFP infected Vero cells; lane 7: uninfected Vero cells
  • Fig. 21 (A) Schematic representation of the immunization schedule. Mice were boosted two weeks after the first inoculation.
  • Fig. 22 Gag-specific CTL response analyzed by 51 Chromium release assay after the 1 st sacrifice, 14 days after priming. Target cells were pulsed with the pool 1 of Gag peptides.
  • FIG. 23 Gag-specific CTL response analyzed by 51 Chromium release assay after the 2 nd sacrifice, 14 days after boosting.
  • the target cells were pulsed with the pool 1 of Gag peptides;
  • the target cells were pulsed with the pool 2 of Gag peptides.
  • Gag peptide pools 1 and 2 are represented in fig. 2 IB
  • Fig. 24 A and B represent respectively INF- ⁇ and IL-4 production post priming in cell-free culture supematants of mice splenocytes after 3 and 6 days of in vitro culture in presence of Gag pool 1 peptides.
  • Fig. 25 A and B represent respectively INF- ⁇ and LL-4 production post boosting in cell-free culture supematants of mice splenocytes after 3 and 6 days of in vitro culture in presence of Gag pool 1 peptides.
  • the data shown in charts correspond to the mean values of different groups of mice.
  • Example 7 Herpetic vector inhibiting angiogenesis and inducing cell suicide in gliomas
  • the present example relates- to anticancer therapy, particularly to a multimodal tumor therapy treatment conseming anti-angiogenic, suicide genes and cytokine genes.
  • the example relates to the use of non-replicate HSV vectors to combine angiogenic inhibitors genes (e.g., angiostatin, endostatin and kringle 5 fusion proteins) with cytokines such as GMCSF or IL12 together with a HSV-tk suicide gene in the same vector to increase their synergistic effects.
  • angiogenic inhibitors genes e.g., angiostatin, endostatin and kringle 5 fusion proteins
  • cytokines such as GMCSF or IL12
  • This strategy will combine three different modalities: (i) the use of angiostatic factors promoting tumor regression along, with (ii) the enzyme-directed prodrug activation (tumor suicide) and (iii) the local production of cytokines, to overcome the inadequate release of tumor antigens and the defect in antigen presentation, and to increase the immune response against the tumor.
  • the last two approaches are devised to enhance the tumor destruction, the inhibition of metastasis and to prevent
  • the method is useful in synergize the effect of cytokines with HSV-TK suicide gene and with antiangiogenesis molecules carried in one He ⁇ es simplex vims based (HSV) vector.
  • HSV He ⁇ es simplex vims based
  • Two angiostatic recombinant vectors have been constracted: one containing the human endostatinXVIII:: angiostatin fusion gene (T0H-endo::angio) and the other containing the human endostatinXVIII::Kringle5 fusion gene (T0H-endo::kringle5), both purchased from InvivoGen (pGT60 and pGT64 plasmids).
  • the genes have been subcloned in EcoRV under the HCMV promoter in a pCDNA shuttle plasmid.
  • the expression cassettes from the derived plasmids pcDNA3 ⁇ NotI/endo-angio e pcDNA3 ⁇ NotI/endo-lringle5 were cut Nrul-Xbal and inserted in Smal-Xbal of a plasmid, named p41, contajLt ⁇ ig the UL41 HSV flanking sequences with HCMN promoter downstream of 100 pb ICPO promoter.
  • the fransgene expression cassettes were inserted into the UL41 locus of HSV genome by replacing the LacZ gene present in the TOZGFP background vector by homologous recombination generating the angiostatic recombinant vectors.
  • the recombinant vimses were identified by isolation of a clear plaque phenotype after X-gal staining. To purify positive isolates we have performed three rounds of limiting dilution. Progeny vimses have been screened by Southern blot analysis to identify recombinants containing the angiostatic genes.
  • PB5 is a pBSSK containing the Usl HSV-1 sequences corresponding to the ICP22 immediate early gene previously described (ref: Krisky DM, et al. Development of he ⁇ es simplex viras replication-defective multigene vectors for combination gene therapy applications.
  • THZ-1 is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMV promoter, in the ICP22 locus.
  • THZ-1 viral DNA and the recombinant plasmid containing the gmcsf gene have been co-transfected into 7b complementing cell line.
  • the recombinant virus TH-gmcsf containing the gmcsf cDNA was identified by isolation of a clear plaque phenotype after X-gal staining.
  • the TH-gmcsf virus was purified by three rounds of limiting dilution and screened by Southern blot analysis to identify the. new recombinant.
  • Western blot analysis and ELISA of the recombinant virus-infected cell lines were performed in order to assess the fransgene expression.
  • the vector T0Hendo::angio/Hgmcsf and T0Hendo::kringle5/Hgmcsf containing the angiostatic fusion proteins in UL41 HSV locus and gmcsf ' in Usl HSV locus was created by genetically crossing the above vectors (T0H:endo::angio or T0H:endo::kringle5 and TH:gmcsf). 7b cells plated on 60 mm petri dishes were infected with 3MOI of T0H:endo::angio or T0H:endo::kringle5 and TH:gmcsf virases and harvested 18 hours post infection.
  • the mixture of viruses derived from the co-infection was titrated, and by Southern blot screening the viral vectors containing both genes were isolated.
  • the T0Hendo::angio Hgmcsf and TOHendo::kringle/Hgmcsf vimses were purified by three rounds of limiting dilution and the genes expression was confirmed by Western blot. analysis.
  • Antiangiogenic fusion proteins were detectable by Western blot in cell culture and supematants of Vero and LLC tumor cells following the infection by T0Hendo::angio and T0Hendo::kringle5 vectors.
  • the media were harvested adding 1 ⁇ g/ml aprotinin.
  • Individual cell monolayers were scraped in Tris-buffered saline (TBS), the cell pellet treated with lysis buffer (0.1 M NaCl, 0.01 M Tris.- HC1 pH 7.0, 0.001 EDTA pH 8.0, 1% Triton X-100, 1 ⁇ g/ml aprotinin, 100 ⁇ g/ml PMSF), sonicated and cleared by cenfrifugation.
  • TBS Tris-buffered saline
  • the proteins were detected with a rabbit polyoclonal antibody specific for the human endostatin (Oncogene Research Products, Boston, USA) 1:1000 diluted and a mouse anti-rabbit IgG PA- conjugated secondary antibody (Promega Co ⁇ oration, USA) at 1:2500 dilution.
  • Antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 2 with TO- GFP recombinant vector.
  • HSV-1 vectors encoding endostatin:: angiostatin (T0Hendo::angio) and endostatin: :kringle5 (T0Hendo::kringle5), as well as TO-GFP control vector were used to infect LLC cells at multiplicity of one. 48 hours later, LLC cell culture supematants were collected from the control and vector-infected cells and tested on human umbilical vein endothelial cells (HUVEC) at different LLC-conditioned vs. normal non conditioned HUVEC medium ratios. HUVEC viability was determined after 5 days by a colorimetric, tetrazolium-based (MTT) assay.
  • MTT colorimetric, tetrazolium-based
  • HUVEC cells were cultured in a 96 well plate (3000 cells/well) and 50 ⁇ l/well of conditioned medium was added. Plates were incubated at 37°C for 5 days and then 25 ⁇ l of MTT (1- (4, 5 dimetiltiazol-2 -il)- 3, 5- dimetilformazane, Sigma M5655) 2,5 mg/m) were added. The precipitate was solubilized with SDS20%-DMF50% (pH 4.7). Absorbance values were measured at 570 nm using a microtifre plate specfrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, UK).
  • ANGIOKIT ZHA- 1000, TCS
  • Human fibroblasts and endothelial cells co-cultured in a 24 well plate constitute the ANGIOKIT system.
  • the co-cultured cells have been infected with the vectors THZ4, TOendo-angio, T0endo-kringle5 e TOendo-angio+TO endo-kringle5 at multiplicity of 1 on days 1, 4, 7 and 10 after cell seeding.
  • Positive and negative controls were freated with recombinant VEGF 2ngr/ml and suramin 20mM, respectively.
  • samples have been fixed with ethanol 70% and then incubated with the primary antibody anti- CD31 and a secondary antibody AP-conjugated.
  • Antibody immunocomplexes were detected by BCIP and NBT substrates in order to show primitive blood vessels (tubes) that had been formed by the endothelial cells. All reagents including the cells were provided by TCS Cell Works 1U4
  • mice 6 weeks old C57B1/6 female mice were injected subcutaneously with 0,5xl0 6 /100 ⁇ l LLC cells and treated with antiangiogenic and suicide genes expressing vectors when the tumors became palpable.
  • the vectors (10 6 plaque forming units/1 OO ⁇ l) were administered every other day, directly into the tumors.
  • the prodrug GCN was injected daily, at lOOmg/kg (i.e. 2,5 mg/mouse in lOO ⁇ l) by intraperitoneal route. Tumor volumes were measured with a digital caliper 3 times a week; each time point represents the average of 6 mice per group.
  • mice injected with LLC infected with T0Hendo::angio + T0Hendo::kringle5 that have shown complete tumor regression were divided in two groups: the follow-up group and LLC-challenge group; the last one has been injected with 1*10 5 LLC in the right flanks. Secondary tumors were 11D treated locally with T0Hendo::angio + T0Hendo::kringle5, in presence or in absence of GCN.
  • the human endostatinXNIII:: angiostatin fusion gene and the human endostatinXVIII::Kringle5 fusion gene both purchased from InvivoGen as pGT60 and pGT64 plasmids, have been cloned in a plasmid containing UL41 HSN flank sequences under the HCMV promoter downstream of lOOpb of ICPO promoter. Both transgenes at the present have been introduced in the same viral locus to avoid differences in gene expression due to promoters and viral location (Fig.26A).
  • TOZ-GFP is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the GFP reporter gene, under HCMN promoter, in the ICP22 locus and the lacZ reporter gene, under ICPO promoter, in the UL41 locus.
  • TOZ-GFP viral D ⁇ A and the recombinant plasmids containing the angiostatic genes have been co-fransfected into a complementing cell line (7b), which provides, in trans, the essential viral genes ICP4 and ICP27.
  • the recombinant viral vector expressing GMCSF was constracted by co-transfecting a plasmid (PB5-gmcsf), which contains gmcsf gene under HCMN promoter flanked by ICP22 HSN1 sequences, with THZ1 recombinant backbone viras into 7b complementing cell line.
  • THZ-1 is a recombinant virus deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMN promoter, in the ICP22 locus (Fig.26B).
  • the vectors TOHendo::angio/Hgmcsf and T0Hendo::l ⁇ ingle/Hgmcsf containing the angiostatic fusion proteins in UL41 HSN locus and gmcsf gene in US1 HSN locus were created by genetically crossing the above vectors (T0H:endo::angio or T0H:endo "kringle and TH:gmcsf) (Fig.26C).
  • Antiangiogenic fusion proteins were detectable by Western blot in cell culture supematants of Nero and LLC tumor cells following the infection by T0Hendo::angio and T0Hendo::kringle5 vectors. In the media, it was evident the release of angiostatic proteins expressed both in the cleaved (20 kDa endostatin) and in the uncleaved form (58 kDa endostatin:: angiostatin and 35 kDa endostatin: :kringle 5) (Fig.27).
  • conditioned media obtained from infected Lewis lung carcinoma (LLC) cells on primary human endothelial cell line (HUVEC) growth was evaluated.
  • Media from LLC (2*10 6 ) cells infected with the recombinant vectors at MOI 2 were collected and overlaid on HUVEC.
  • bFGF 3ngr/ml was added as angiogenic stimulus.
  • Negative confrols were represented by HUVEC cells treated with medium obtained from non-infected LLC or LLC infected with the confrol vector (THZ4). After 5 days incubation HUVEC viability was determined by a colorimetric, tetrazolium-based (MTT) assay. All samples were run in quadruplicate (Fig.28).
  • HUVEC recombinant human antiangiogenic proteins
  • mice injected with LLC infected with T0Hendo::angio + T0Hendo::kringle5 which have shown complete tumor regression were divided in the follow-up (A) and LLC-challenge (B) groups. Secondary tumors were treated locally with T0Hendo::angio + T0Hendo::kringle 5, in presence or in absence of GCV. Mice with rapidly growing tumors present significant spleen enlargement, also observed in the pre-established tumor treatment-model (Fig.34).
  • a DNA fragment comprising an HSV ICPO-immediated early promoter-lacZ expression cassette flanked by Pad restriction endonuclease recognition sites and sequences homologous to UL41 for HSV-1 was co-transfected in 7b cells with HSV-1 mutant. Recombinants were screened for LacZ expression, and the correct insertion was confirmed with Southern blot analysis.
  • the resulting recombinant viras was used, as backbone, to construct the vimses to use in an anticancer therapy.
  • the recombination was designed to remove the LacZ expression cassette from the vimses to be substituted with expression cassettes containing the HIV gene either under ICPO-IEp promoter or HCMV-IEp promoter.
  • isolates were screened with X-gal staining, and white plaques were selected and screened by Southern blot hybridization using a probe hybridizing the specific gene (e.g., endostatin, angiostatin, kringleS, gm-csfi etc.).
  • a probe hybridizing the specific gene e.g., endostatin, angiostatin, kringleS, gm-csfi etc.
  • Vectors containing more then one non-native expression cassette were obtained by genetic crosses between the recombinant vimses. Such manipulations are known in art.
  • ICP4 " , ICP27 " , and ICP22 " HSV vectors having an ICP4-immediate early promoter-TK expression cassette at the UL24 locus are known (Marconi, P., Krisky, D., Oligino, T., Poliani, P.L., Ramakrishnan, R., Goins, W.F., Fink, D.J., & Glorioso, J.C. (1996). Replication-defective he ⁇ es simplex vims vectors for gene transfer in vivo. Proc Natl Acad Sci USA, 93(21), 11319- 11320.) The replication defective HSV-1 vectors are modified by having non-native expression cassettes encoding for HIV proteins at the UL41 or ICP22 locus.
  • T0Hendo::angio and TOHendo "kringle 5 viral vectors express the suicide HSV-TK gene and are able to kill the infected cells in presence of GCV in vitro;
  • TOHendo ::angio and TOHendo "kringle 5 are able to exert significant inhibitory activity on proliferation and migration of human endothelial cells in vitro, as well as tubes formation in mixed cultures of endothelial cells and fibroblasts; treatment of pre-established LLC tumors in vivo with TOHendo ::angio and TOHendo: :kringle 5 vectors is capable of reducing the tumor growth rate, but is not able to completely arrest it; antitumoral effect is further increased by systemic adminisfration of GCV; infection of LLC tumor cells with TOHendo ::angio and TOHendo:.
  • FIG.26 Schematic representation of recombinant vectors
  • .-angiostatin (TOHendo: :angio) and endostatin: :kringle 5 (TOHendo "kringle 5) genes were cloned in the UL41 locus of the TO-GFP backbone vector, both under the HCMV promoter; HSV-1 thymidine kinase (TK) gene is located in its natural UL23 locus, under ICP4 IE (instead of the native early) promoter control.
  • TOHendo :angiostatin
  • TK thymidine kinase
  • Murine gmcsf gene under the HCMV promoter, has been cloned in ICP22 locus of THZ.1 backbone vector.
  • FIG. 27 In vitro expression of antiangiogenic proteins
  • Lanes 8, 9 and 10 show MW marker, recombinant human endostatin (positive confrol) and uninfected LLC cell culture medium, respectively.
  • FIG.28 In vitro biological activity of antiangiogenic proteins: effect on endothelial cell proliferation
  • HSV-1 vectors encoding endostatin:: angiostatin (TOHendo:: angio) and endostatin: :kringle 5 (TOHendo: :kringle 5), as well as THZ.4 confrol vector were used to infect LLC cells at multiplicity of one. 48 hours later, LLC cell culture supematants were collected from the confrol and vector-infected cells and tested on human umbilical vein endothelial cells (HUVEC) at different LLC-conditioned vs. normal non conditioned HUNEC medium ratios. HUNEC cell proliferation was evaluated after 5 days by MTT assay.
  • FIG. 29 In vitro biological activity of antiangiogenic proteins: effect on endothelial cell migration.
  • FIG. 30 In vitro biological activity of antiangiogenic proteins: effect on tube formation in primary human endothelial + fibroblast co-cultures.
  • FIG. 31 Cytotoxic activity of HSV-1 TK suicide gene following prodrag Ganciclovir (GCV) addition to the cell culture media.
  • Charts A, B cytotoxicity of TK + antiangiogenic factors (TOHendo:: angio and TOHendo "kringle 5) -expressing vectors in presence (A) and in absence (B) of Ganciclovir on LLC tumor cells; charts C, D: cytotoxicity of TK + antiangiogenic factors (T0Hendo::angio and TOHendo: :kringle 5) -expressing vectors in presence (C) and in absence (D) of Ganciclovir on primary endothelial HUNEC cells.
  • FIG. 32 In vivo local treatment of pre-established LLC tumors with the recombinant TK and antiangiogenic proteins- expressing vectors.
  • mice 6 weeks old C57B1/6 female mice were injected subcutaneously with 0,5xl0 6 LLC cells and treated with antiangiogenic and suicide genes expressing vectors when the tumors became palpable.
  • the vectors (10 6 plaque forming units/lOO ⁇ l) were administered every other day, directly into the tumors.
  • the prodrag GCN solution was injected 24 hours (chart A) or 48 hours (chart B) post viral injection and administered daily, at lOOmg/kg (i.e. 2,5 mg/mouse in lOO ⁇ l) by intraperitoneal route.
  • Tumor volumes were measured with a digital caliper 3 times a week; each time point represents the average of 6 mice per group.
  • FIG. 33 In vivo tumor growth inhibition by implantation of vector-infected LLC cells LLC (1*10 6 cells) were infected at multiplicity of 1 with the recombinant viral vectors, and then injected into the left flanks of C57B1/6 mice. Ganciclovir (lOOmg/kg) was administered i.p. for a week afterwards. Tumor volumes were ' measured with a digital caliper every two days; each time point represents the average of 12 mice per group. Mice injected with LLC infected with TOHendo:: angio + TOHendo ::kringle5 have shown complete tumor regression. Images show representative primary tumor-injection sites (left flanks) of mice belonging to different groups at the time points indicated by the black arrows.
  • FIG. 34 In vivo follow up and tumor challenge growth inhibition by implantation of vector- infected LLC cells.
  • mice injected with LLC infected with TOHendo:: angio + TOHendo: :kringle5 that have shown complete tumor regression were divided in the follow-up (A) and LLC-challenge (B) groups.
  • secondary tumors were treated locally with TOHendo:: angio + TOHendo: :kringle5, in presence or in absence of GCN.
  • Representative images of secondary tumors (challenge; right flanks) are shown in (B) chart, together with the spleens. Mice with rapidly growing tumors present significant spleen enlargement, also observed in the pre- established tumor treatment-model.
  • Example 8 Gene Therapy For Nervous System : Defective Hsv Vector Expressing Fgf2 And Bdnf And Synergic Effect On In Vitro Proliferation And Differentiation
  • This Example realtes to the use of non-rep licate HSV vectors: (i) to treat acquired Neurological Diseases such as Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis in the CNS and in the peripheral nervous system (PNS) by combining neurotrophic factors genes together in the same vector to increase their synergistic effects or (ii) to treat Neurodegenerative diseases with genetic involvement such as Tay-Sachs Disease (TSD) by expressing a missing enzyme.
  • acquired Neurological Diseases such as Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis in the CNS and in the peripheral nervous system (PNS) by combining neurotrophic factors genes together in the same vector to increase their synergistic effects
  • PNS peripheral nervous system
  • TSD Tay-Sachs Disease
  • the CNS with its unique complex structures consisting of a variety of cell types and tracts kept in a privileged environment separated from the blood system by the blood-brain barrier (BBB) represents a barrier to physiopatholocical studies and to gene therapy.
  • BBB blood-brain barrier
  • Neurotrophins represent a large family consisting of nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotro ⁇ hin-3,-4,-5, and 6 and glial-derived ' neurotrophic factor (GDNF).
  • NGF nerve-growth factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glial-derived ' neurotrophic factor
  • Neurotrophins affect a large number of neuronal cells in developing and adult neuronal populations of the peripheral nervous system (PNS) and CNS and play important roles in proliferation and differentiation of cortical progenitorscells to a particular lineage.
  • PNS peripheral nervous system
  • neurotrophins can provide neuroprotection to neurons axotomized by traumatic injury or by neurotoxins.
  • Two other growth factors involved in progenitor cell proliferation and in graded stages of neuronal differentiation belong to the cytokines family and are the basic fibroblast growth factor (bFGF or FGF-2) and the ciliary neurotrophic factor
  • HSN he ⁇ es simplex viras
  • Replication deficient HSN-1 based vectors have several characteristics which make them particularly attractive for gene transfer to neurons: (a) HSN-1 has an intrinsic neurotropism; b) vectors can be grown to high titer without the generation of replication competent viras (c) second generation vectors have low cytotoxicity for neurons; (d) replication-deficient viruses have been demonstrated to establish latency in the cell body of motor neurons following inoculation of muscles in the extremities of animals; (e) HSV contains an endogenous promoter system which has been shown to remain active long-term albeit low levels in neurons; (f) vectors can be engineered to contain multiple transgene expression cassettes.
  • Vero cells African green monkey kidney cells from ATCC, Rockville, MD, USA; CCL81
  • Vero-derived cell line, termed 7b that expresses the HSV-1 immediate early genes ICP4 and ICP27 required for viras replication
  • ICP4 and ICP27 required for viras replication
  • Oligodendrocyte-type 2 astrocyte precursor cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with NI supplement (1 ml/100 ml, Sigma), penicillin/streptomycin 2%, L-glutamine (1 ml 100 ml) and biotin (1 g/100 ml, Sigma).
  • DMEM Dulbecco's Modified Eagle's Medium
  • the first one contained the sequences for the rat ovarian basic fibroblast growth factor (FGF-2) cDNA (A. Baird, The Whittier Institute, La Jolla, CA, USA) were placed downstream of the HCMV IE promoter and 5' to the polyadenylation sequences from the bovine growth hormone.
  • the second construct contained the Escherichia Coli LacZ gene driven by the HCMV immediate early promoter (THZ vector).
  • Plasmid pB410-BDNF was constructed by introduction of the rat brain-derived neurotrophic factor (bdnf) cDNA (pb) from pBluscript-BDNF (kindly given by Marco Riva, Milan) into the HSV flank sequences .of pB41 plasmid that has been described elsewhere (ref: Krisky, D.M., Wolfe, D., Goins, W.F. et al. Deletion of multiple immediate-early genes from he ⁇ es simplex viras reduces cytotoxicity and permits long-term gene expression in neurons. Gene Ther 1998, 5 (12), 1593-1603.).
  • the bdnf cDNA under the transcriptional control of the HSV immediate-early ICPO promoter was inserted into Xbal sites of pB41 plasmid between the two UL41 HSV fragments (HSV genomic positions 90.145-91.631 and 92.230-93.858) (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C (1997). Rapid method for constraction of recombinant HSV gene fransfer vectors. Gene Therapy 4:1120-1125.).
  • Plasmid pB410-BDNF was recombined within the genome of the TOZGFP viral vector using the previously described Pac-facilitated lacZ substitution method (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C. (1997). Rapid method for constraction of recombinant HSV gene fransfer vectors. Gene Therapy 4:1120-1125.).
  • the TOZGFP is a replication-defective HSV-1 viral vector this vector has low toxicity due to the deletion of three immediate early genes (ICP4, ICP27, which are essential for viral replication and ICP22) and contains the gfp (green fluorescence protein) gene into the ICP22 locus and the LacZ gene in the UL41 locus as marker genes.
  • ICP4, ICP27 immediate early genes
  • gfp green fluorescence protein
  • the production of recombinant virases was carried out using the standard calcium phosphate transfection procedure with 5 ⁇ g of viral DNA and 1 ⁇ g of linear plasmid pB410-BDNF. Transfection and isolation of the recombinant virus was performed in 7b cells as previous described.
  • the recombinant viras T0:BDNF/GFP containing the bdnfcDNA was identified by isolation of a clear plaque phenotype after X-gal staining.
  • the T0:BDNF/GFP viras was purified by three rounds of limiting dilution and the presence of the fransgene was verified by Southern blot analysis.
  • Viral stocks of the TO: BDNF/GFP and of the control vector TO-GFP (derived from TOZGFP without lacZ reporter gene in UL41 locus) were prepared and titrated using Vero- 7b cells (ref: Marconi, P., Krisky, D., Oligino, T. et al. Replication-defective he ⁇ es simplex vims vectors for gene transfer in vivo. Proc Natl Acad Sci USA 1996, 93(21), 11319-11320).
  • the vector TH:FGF/0:BDNF/GFP containing FGF-2 in Tk HSV locus and BDNF in UL41 HSV locus was created by genetically crossing the above vectors (TH-FGF2 and T0:BDNF/GFP).
  • 7b cells plated 60 mm petri dishes were infected with 3MOI of TH:FGF and TO: BDNF virases and harvested 18 hours post infection.
  • the mixture of virases derived from the co-infection was titrated, and the viral vector containing both genes was isolated by Southern blot screening.
  • the TH:FGF/0:BDNF/GFP viras was purified by three rounds of limiting dilution and the genes expression were confirmed by Western blot analysis.
  • Vero cells were plated in 6-well plates (2 10 5 cells/well). One day after plating, some wells were infected at a MOI of 1.0 with TH:FGF-2, T0:BDNF/GFP, TH:FGF/0:BDNF/GFP, THZ, TOZ. One to seven days after infection, individual cell monolayers were scraped in Tris-buffered saline (TBS), the cell pellet treated with lysis buffer (0.1 M NaCl, 0.01 M Tris.-HCl pH 7.0, 0.001 EDTA pH 8.0, 1% Triton X-100, 1 g/ml aprotinin, 100 g/ml PMSF), sonicated and cleared by centrifugation. The presence of FGF-2, BDNF and FGF/ BDNF was determined by Western blot and ELISA assays.
  • FGF and BDNF protein expression from the TH:FGF-2, T0:BDNF/GFP, TH:FGF/0:BDNF/GFP vectors was analyzed in Vero cells (lxlO 6 cells) infected with 1 MOI of the respectively virases. Cell exfracts, corresponding to 10 g of total proteins, were ran onto 12% SDS-polyacrylamide gel and analyzed by western blot. The FGF protein was revealeded using a mouse anti-basic human FGF monoclonal antibody (Transduction Laboratories, Lexington, Kentucky) at 1:250 dilution and a mouse anti-mouse HRP-conjugated secondary antibody (NA931V, Amersham) at 1:2500 dilution.
  • a mouse anti-basic human FGF monoclonal antibody Transduction Laboratories, Lexington, Kentucky
  • a mouse anti-mouse HRP-conjugated secondary antibody NA931V, Amersham
  • Immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech).
  • the BDNF protein was revealeded using a polyclonal rabbit anti human BDNF (S.Cmz, SC546) 1:500, which reacts also with rat BDNF and a detected using an anti-rabbit IgG HRP- conjugate (NA934V, Amersham) l:250.0.and TMB substrate.
  • Controls were represented by uninfected cells and cells infected with 1 MOI of the THZ or TOZGFP recombinant vectors.
  • FGF-2 R&D System, Minneapolis, MN, USA
  • BDNF BDNF
  • Brains were minced and digested in 10 ml HBSS with 0.0025% frypsin/EDTA (Gibco). After 20 min at 37°C, 5 ml fetal bovine serum (FBS, to stop trypsin action) and 30 ml HBSS were added and the whole was centrifuged for 10 min at 1,500 r.p.m. The supernatant was discarded and the digested tissue was mechanically dissociated by two passages through cell strainers (mesh size 100 micrometer and 70 micrometer). Fresh HBSS was added to help cells through, reaching a final volume of 32 ml. Dissociated cells were layered on 4 preformed Percoll (Sigma) gradients.
  • FBS fetal bovine serum
  • Percoll is a density gradient material consisting of colloidal silica coated with polyvinylpyrrolidone. Percoll gradients are suitable to sediment the cells in isopycnic banding. Cells are thus separated according to buoyant density. The density of cells and subcellular particles is a function of their water content and is very sensitive to the colloid osmotic pressure, pH and ionic strength. Progenitor cells are separated rapidly from postmitotic cells using density gradients because they have densities higher than differentiated cells.
  • the solution was made by mixing 9 ml Percoll, lml HBSS/10X, 10ml HBSS/1X and was centrifuged at 14,000 r.p.m. for 30 min at 4°C using swing-out rotors. The cell suspension was then centrifuged at 14,000 t.p.m. for 15 min at 4°C After centrifugation, the material was fractionated in four gross fractions.
  • fractions were: a) a clear supernatant, b) a white, thin, myelin-like layer, c) a large, slightly opalescent fraction, and d) a layer of red blood cells on top of a relatively dense-appearing pellet.
  • Fractions a and b were vacuum aspirated and eliminated.
  • Fraction c i iy consisted of the enriched progenitor fraction.
  • DMEM Dulbecco's Modified Eagle's Medium
  • NI supplement (1 ml/100 ml, Sigma)
  • penicillin/streptomycin 2% penicillin/streptomycin 2%
  • L- glutamine (1 ml 100 ml
  • biotin (1 g/100 ml, Sigma).
  • the purified cell population obtained consisted of.oligodendrocyte-type 2 astrocyte precursor cells and neurons precursors in the form of steady, proliferating homotypic aggregates called "neurospheres”.
  • the purified cell population was seeded at high cell density (1.5xl0 5 cell/cm 2 ) in uncoated plastic tissue culture flasks (T25) and fed with DMEM-N1 (supplemented as described above) and 2% fetal bovine serum (FBS). Flasks were kept at 37°C in a humidified 9% CO 2 /91% air atmosphere for 72 hr. After elimination of adhering cells, the neurospheres were disaggregated by gentle trituration with a micropipet and the cells were counted in a hemacytometer.
  • the progenitor cells were resuspended in aliquots of 5x10 5 cells and these aliquots were infected with 5xl0 5 pfu TH-lac Z, TH-FGF2, TO- BDNF/GFP or TH-FGF2/0- BDNF/GFP recombinant vimses (i.e., at a MOI of 1.0).
  • Infected cells were incubated at 37°C for 30 min, resuspended in serum-free DMEM, layered in 100 1 droplets onto glass coverslips precoated with 100 g/ml polyomithine (posed into 12-well tissue culture plates) and allowed to attach for 1 hr before adjusting the medium to the final volume of 2 ml per well. Finally, cells were cultured in serum- free DMEM without other growth factors for 4 weeks, to dissect out the effects of the neurotrophic factors produced by the vectors.
  • coverslips were washed twice with PBS for 10 min, incubated with TR-conjugated secondary goat anti-mouse antibody (1:250) for 45 min at room temperature, and washed three times with PBS. The last wash contained 10 ng/ ml 4', 6-diamidino-2-phenylindole (DAPI, Sigma) in PBS. This was used as a fluorescent counterstain for cell nuclei. Coverslips were mounted using Gel/ MountTM (Biomeda).
  • FIG. 35 A schematic representation of the vectors employed in this study is shown in Fig. 35. All these vectors are triple mutants, defective for the ICP4, ICP27 and ICP22 immediate-early genes, that ensure efficient expression of the fransgene. Although this mutant background contains some degree of residual toxicity for some cell types, this phenomenon does not appear to be long- lasting nor to heavily affect neurons.
  • FGF-2 For FGF-2, we used a previously characterized vector, TH- FGF2, in which the fransgene was introduced into the tk (UL23) locus of the control (TH-lacZ) vector by digestion of the viral DNA and by homologous recombination with an expression cassette containing sequences coding for FGF-2 under the confrol of the human cytomegalovirus immediate-early (HCMV IE) promoter.
  • HCMV IE human cytomegalovirus immediate-early
  • BDNF For BDNF, we used a vector, TO- BDNF/GFP, in which the transgene was introduced into the UL41 locus of another confrol vector (TO-lacZ/GFP) by digestion of the viral DNA and by homologous recombination with an expression cassette containing sequences coding for BDNF under the control of the ICPO promoter.
  • the rationale for choosing these two distinct promoters was to mimic a sequential activation of FGF-2 and BDNF: in fact, the HCMV IE promoter ensures robust, but very transient, transgene expression, while the ICPO promoter provides a longer-lasting expression.
  • the double mutant (TH-FGF2/0- BDNF/GFP) was obtained by crossing over TH-FGF2 and TO- BDNF/GFP (Fig.35). TH-lacZ and TO-lacZ/GFP were used as controls.
  • ELISA assays were performed on total proteins extracted from Vero cell monolayers infected with the different vectors at a multiplicity of infection (MOI) of 1.0. Vero cells were infected with TH-FGF2, TO-BDNF/GFP, TH- FGF2/0:BDNF/GFP or their respective confrols (TH-lacZ and TO-lacZ/GFP), and FGF-2 or BDNF expression was determined at different times post-infection (1 to 4 days).
  • MOI multiplicity of infection
  • type 2 asfrocytes were identified on the basis of expression of GFAP (glial fibrillary acid protein); neurons on MAP2 (microtubule-associated protein-2); oligodendrocytes on CNPase (2',3'-cyclic nucleotide-3' phosphodiesterase).
  • the cells were also checked for the infection of HSV-1 based vectors with the polyclonal antibody anti HSV As described above, confrol cells and cells infected with the control vector TH-lacZ or the FGF-2 expressing vector TH-FGF2 or the BDNF expressing vector TO- BDNF/GFP differentiated in different proportion in a mixed culture containing GFAP-positive cells (presumably asfrocytes), CNPase- and MAP2-positive cells (presumably oligodendrocytes and neurons) (Fig.39) The vector expressing FGF-2 and BDNF together (TH-FGF2/0- BDNF/GFP) proliferate and differentiated in a significant proportion in neurons (Fig. 40 A and B).
  • the present data demonstrate the feasibility of use of HSV-1 vectors for obtaining long-term biological effects in an in vivo system; extend previous observations that synergies occur between different NTFs, by showing a synergistic effect between FGF-2 and BDNF, and indicating that this occurs at the single cell level: in fact, the insertion of expression cassettes for both NTFs in the same viral backbone ensures transfection of both genes of interest in every infected cell; suggest that it will be possible to manipulate CNS cell proliferation, differentiation and migration by using appropriate combinations of NTFs: if achieved, such results would form the basis for the development of new strategies for the gene therapy of neurological disorders characterized by or associated with loss of specific CNS cell types.
  • FIG. 35 Graphic map of the viras TH-FGF2/0-BDNF/GFP, a triple mutant containing the rat FGF-2 gene in the tk locus, the rat BDNF gene in the UL41 locus, and the GFP cDNA in the ICP22 locus.
  • the FGF-2 and GFP genes are driven by the HCMV IE promoter; the BDNF gene is driven by ICPO IE promoter.
  • FIG. 36 Time course of BDNF expression in vitro as determined by ELISA.
  • the NTF expression was assayed on clarified lysates from TOZ-GFP, and TO-BDNF/GFP infected Neuro2A cells (1 pfu/cell) using an anti-human BDNF antibody. Cells were harvested every 24 h up to 3 days
  • FIG.37 A Western blot analisys for BDNF: by infecting at moi 1 Vero cells with TH- FGF2/0:BDNF/GFP, TOBDNF (as positive control) and TOGFP (as negative control).
  • Primary antibody Polyclonal rabbit anti human BDNF (S.Cruz, SC546) 1:500, which reacts also with rat BDNF; secondary antibody: antirabbit IgG HRP- conjugate (NA934V, Amersham) 1 :2500.
  • TOGFP infected Vero cells 24h post infection; lane 2: MW marker; lanes 3-5: TH- FGF2/0:BDNF/GFP Vero infected cells (three vectors); lane7: THFGF Vero infected cells; lane 8: non-infected Vero cells; lane9: recombinant human BDNF protein (Promega, G1491), 50ngr.
  • FIG.38 Schematic representation of the progenitore maturation ant the cellular markers that are acquiring during the differentiation in the diffemt lineages
  • FIG.39 Graphic representation of proliferation and differentiation of progenitor cells infected with the different viral vectors.
  • FIG. 40 In vitro differentiation of progenitors infected with TH-FGF2/0:BDNF/GFP vector expressing FGF-2 and BDNF together.
  • Cellular immunofluorescence experiments performed progenitors isolated from the newborn rat CNS infected with TH-FGF2/0:BDNF/GFP and maintained in minimum essentail, serum-free DMEM medium for 4 weeks. Neurons were identified on the basis of expression of MAP2.
  • Example 9 A direct gene transfer strategy via brain internal capsule delays the progression of Tay-Sachs disease and revert the altered phenotype
  • TS Tay-Sachs
  • HexA ⁇ -hexosaminidase A
  • MUG 4-methylumbelliferyl ⁇ -N-acetylglucosaminide
  • MUGS 4-methyumbelliferyl ⁇ -N-acetylglucosamine-6-sulfate
  • Hexosaminidase Hex
  • HSN- TOalphaHex Hexosaminidase
  • Tay-Sachs disease is a GM2 gangliosidosis due to the deficiency of the ⁇ - subunit of ⁇ -hexosaminidase A l ' 2 (HEXA gene).
  • HEXA gene ⁇ -hexosaminidase A l ' 2
  • Pathological features include wide neurodegeneration and neuronal lipid storages.
  • TS treatment is restricted to supportive care and appropriate management of intervening problems 3 .
  • Hex A ⁇ structure
  • Hex B ⁇ structure
  • the homodimer ⁇ Hex S
  • ⁇ ⁇ structure
  • Hex S represents the residual Hex activity in Sandhoff disease patients, a type 0 GM2 gangliosidosis due to inherited defects in the HEXB gene 4"7 .
  • Hex A in the presence of the GM2-activator protein, hydrolyses the ⁇ -GalNAc-(l-4)- ⁇ -Gal glycosidic linkage of the GM2 ganglioside 1 ' 7"10 .
  • CNS Central Nervous System
  • Several therapeutic approaches allow restoring the enzymatic activity in many key tissues (kidney, liver, spleen, etc.) but the reduction of the GM2 ganglioside deposits in the CNS is difficult to achieve 11"16 .
  • CNS is kept in a privileged environment, separated from the blood system by the blood-brain barrier (BBB), that represents an obstacle to therapy.
  • BBB blood-brain barrier
  • a further obstacle stems from the observation that the fibroblasts from TS patients are not cross- corrected in vitro by simply adminisfration of the missing enzyme 17 and hider recent data supporting the concept that therapeutic approaches that heighten expression of the lacking enzyme in macrophages/microglia serves to enhance the corrective therapeutic potential 18,19 .
  • TS pre-clinical diagnosis is an as fortunate as sporadic event, a therapeutic approach could be effective if it is able to delay the acute phase of the disease. Therefore restoration of Hex A activity and reduction of GM2 ganglioside storage represent one of the first major goal.
  • RESULTS Construction of herpes simplex viral vector encoding for Hex -subunit We produced the non- replicating he ⁇ es simplex viral vector containing the human Hex ⁇ -subunit cDNA under the control of the ICPO promoter (HSV-TO ⁇ Hex) (Fig.41a) 24"25 .
  • ICPO promoter that is an immediate early viral promoter, is up-regulated to extremely high levels in a background of a triple mutant replication-deficient vector.
  • the expression cassette, containing the cDNA surrounded by UL41 flanking sequences of HSV, has been recombined into UL41 locus of the
  • HSV-TO ⁇ Hex therapeutic viral vector
  • HSV-TOZ control viral vector
  • mice 4 weeks after the injection, we sacrificed the mice, separated the brain hemispheres, the cerebellum and the spinal cord. We dissected each brain hemisphere in 4 rosfro-caudal 2.5 mm thick sections and the spinal cord in 3 antero-posterior segments. We analyzed each brain section, as well as cerebellum and spinal cord, for Hex A activity and for GM2 ganglioside content.
  • HSV-TOZ viral vector Distribution of the HSV-TOZ viral vector in treated TS mice.
  • mice were evaluated the efficacy of our strategy by injecting the animal model of TS disease 18 with the HSV-TO ⁇ Hex.
  • TS mice express a mild form of the disease 19 ' 29 . They do not show neurologic symptoms, however they are lacking in Hex A activity (Fig.42) and present storage of GM2 ganglioside in the brain (Fig.43). Therefore these mice represent a suitable experimental model for testing the effectiveness of our approach on slowing the progression of the disease.
  • the lower level of the Hex A activity was sufficient to hydrolyze the GM2 ganglioside but it was not sufficient to completely remove the lipid storage. We hypothesized that either it is need a grater number of cells that express the enzyme or the lower viral dose could need a longer time to completely remove the storage material.
  • the animal model of TS disease was generated by Yamanaka et al. (18) and bred in our laboratory. C57/bl6 mice were from Charles-River, Italy.
  • Bovine seric albumin and Bio-Rad protein assay reagent were from Bio- Rad Laboratories, DE-52 DEAE-Cellulose was from Whatman Biochemicals. Thin-layer 20x20 CM plates TLC, were form E.Merck A.G. (Darmstadt, Germany). The medium for tissue culture was from Euro-Clone, Celbio Lab., fetal calf serum was from Mascia Brunelli, penicillin/streptomycin was from Gibco BRL. All other reagents were of analytical grade.
  • the non-replicating HSV-1 viral vector which expresses the ⁇ -subunit cDNA of Hex A, has been created using the Pad recombination system according to the methods of Krisky 26 .
  • the ⁇ -subunit cDNA of Hex A was cloned under the transcriptional control of ICPO promoter.
  • TOZ vector derived from the replication-deficient T.l, defective for ICP4, ICP27, ICP22 immediate early genes and for UL41 gene where the ⁇ -Galactosidase cDNA, under the control of the ICPO IE promoter and flanked by Pad restriction sites (which are not present into the viral genome), was inserted into the vhs (UL41) locus.
  • Potential recombinant viras was identified by a "clear plaque" phenotype after the X-Gal staining since the insertion of the transgene constract into the viral genome have eliminated the LacZ gene.
  • the TO ⁇ Hex recombinant vims was purified by three rounds of limiting dilution and verified by Southern Blot analysis for the presence of the ⁇ - subunit cDNA of Hex A and for the correct insertion of the named gene in the viral genome.
  • TS organotypic brain slices preparation and transduction with the HSV-TOaHex were produced and cultured under standard condition defined in Malgaroli's laboratories. For each experiment, at least three animals and 18 slices were used. After decapitation, 5 months-old TS and wild-type mice (C57/bl6) brains were dissected out into cold Gey's balanced salt solution containing 5 mg/ml glucose. Brain coronal slices (500 ⁇ m thick) were cut on a Mcllwain tissue chopper and transferred into membranes of 30 mm Millipore culture inserts with 0.45 ⁇ m pore size (Millicell; Millipore, Bedford, MA).
  • the medium was composed of 50% basal medium with Earle's salts (Invitrogen, Gaithersburg, MD), 25% HBSS (Invitrogen), 25% horse serum (Invitrogen), L- glutamine (1 mM), and 5 mg/ml glucose.
  • TOal ⁇ ex direct injection into the brain internal capsule of TS mice.
  • Five groups of 5 months old animals were injected with two different doses of TO ⁇ Hex into the internal capsule of the left-brain hemisphere of the TS mice.
  • Mice were anesthetized with 0.02 ml/g body weight of 2,2,2-tribromoethanol and 2-methyl-2-butanol and placed on the Styrofoam platform of a stereotaxic injection apparatus (David Kopf Instruments, Tujunga, California, USA). The skull was exposed by a 10-mm incision in the midline.
  • the injection coordinates for the internal capsule were -0.34 mm to bregma, 1.4 mm mediolateral, and.3.8 mm of depth.
  • Each injection was 5 ⁇ l total, and the injection speed was 0,l ⁇ l/min.
  • the injections were carried out using a needle capillary (1.2 mm x 0.6 mm) attached to a Hamilton syringe. The injections were delivered at a rate of O.l ⁇ l/min, and the needle was slowly withdrawn after an additional 5 minutes. The scalp was closed by suture.
  • HSV-TOZ viral vector distribution One month after injection some mice were sacrificed by cardiac perfusion. The left ventricle was cannulated, an incision was made in the right atrium, and the animals were perfused with 2% paraformaldehyde in PBS until the outflow run clear then the brain included in ornithyne carbamoyl fransferase (O.C.T. TM Compound TISSUE- TEK, Sakamura, The Netherlands) after exposure at 5% - 30% glucose gradient and finally sectioned on a cryostat into 15 ⁇ m thick serial sections). ⁇ -Gal positive cells were assayed through X-Gal staining (39) . Animal experimentation protocols were approved by the HSR Institutional Animal Care.
  • Brain extracts At sacrifice some mice were decapitated and the brain hemispheres dissected in 4 rosfro-caudal 2,5 mm sections and cerebellum. Organotypic brain slices and brain sections, were homogenized in a Potter Elveheim type homogenizer in lOmM-sodium phosphate buffer, pH 6.0, containing 0.1 % (v/v) Nonidet NP40 detergent and sonicated. The lysates were centrifuged at 12,000 ⁇ m Eppendorf microfuge for 20 min and supematants used as tissue extracts for enzyme analysis. All procedures were carried out at 4°C
  • ⁇ -Hexosaminidase activity assay Enzyme activity was determined by using two fluorogenic substrates: 3 mM MUG or MUGS in 0.1 M- mecanicte/0.2 M-disodium phosphate buffer at pH 4.5 ' ' . Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin Elmer LS3 fluorimeter (excitation 360 nm, emission 446 nm). ⁇ -Hexosaminidase isoenzymes analysis. Tissue extracts were analyzed by the ionic-exchange chromatography on DEAE-cellulose .
  • the chromatography was performed by using 1 ml column equilibrated with 10 mM-Na phosphate buffer, pH 6.0 (buffer A). The flow rate was 0.5 ml/min. Enzyme activity retained by the column was eluted by a linear gradient of NaCl (0.0-0.5 M in 40 ml of buffer A). Finally, the column was eluted with 1.0 M-NaCl in the same buffer. Fractions (1ml) were collected and assayed for the Hex activity.
  • Gangliosides extraction and quantitative determination were extracted from mouse brain and cerebellum (10-70 mg of tissue) by using the method of Folch 40 as modified by Hess and Rolde 41 . Briefly, the weighted frozen tissue was thawed, manually homogenized in a 1 ml potter-Elvehjem homogenizer with Teflon pestle and to the mash was added 2:1 chloroform- methanol mixture (v/v). in a volume 20 fold the tissue weight. The obtained homogenate was centrifuged 10 minutes at 5000 ⁇ m in an Eppendorf Centrifuge 5415D and the supernatant was recovered. This cmde extract was partitioned by adding 20% of its volume of re-distilled water and mixing.
  • the two phase were separated by centrifugation for 15 minutes at 3000 ⁇ m in a microfuge: the upper phase was accurately recovered and the interface rinsed with a few tenths ⁇ l of theoretical upper phase (3:48:47 chloro form-methanol- water); the lower phase was re- extracted with a volume of theoretical upper phase containing 0.015 M of KC1 in water. After centrifugation, the recovered upper phase was combined with the first one and dried.
  • the total ganglioside concentration was determined by resorcinol-HCl method according to Svennerholm with 85:15 butyl acetate-butyl alcohol as extiactant 42 and added to an eppendorf tube containing an equal volume of appropriately diluted upper phase re-suspended in water, mixed well, and heated at 100 °C in a thermo-bloc for 15 minutes. After addition of 1 ml of extiactant and mixing, the samples were cooled on ice water and centrifuged for 3 minutes at 5000 ⁇ m. The solvent layer was recovered and measured at 580 nm wavelengths using a Shimadzu UV- Visible Recording Specfrophotometer (UV-160A). The sialic acid concentration was determined by comparison with a standard curve.
  • Fig.41 Recombinant HSV-1 non-replicating viral vector containing Hex ⁇ -subunit cDNA.
  • One unit (U) is the amount of enzyme that hydrolyses 1 ⁇ mol/min of substrate at 37°C ⁇ TS, Tay-Sachs mice; E ⁇ DTS- TO ⁇ Hex, Tay-Sachs mice transduced with the HSV-TO ⁇ Hex; I WT, C57 B16 mice; 0 TS-T0Z, Tay-Sachs mice transduced with the HSV-TOZ.
  • Fig.42 Hex A activity in TS mice brain transduced with HSV- TO ⁇ Hex viral vector.
  • Hex activity was assayed towards the synthetic substrate MUGS in brain section exfracts a) and cerebellum b); LI, L2, L3, L4 and RI, R2, R3, R4 are the left and right brain hemisphere sections respectively.
  • Fig.43 Decrease of GM2 ganglioside in TS brain sections transduced with HSV-TO ⁇ Hex viral vector.
  • Fig. 44 HSV-TOZ distribution into the mouse central nervous system.
  • Serial brain sections were produced dissecting animals in coronal, transversal and sagittal orientation. Sections were stained with the X-Gal substrate, a) Coronal brain sections; al-a9: representative coronal serial sections; a4: site of injection (IC, internal capsule) b) Transversal brain sections; bl-b8, representative transversal serial sections c) Sagital brain sections; cl-c8, representative sagittal serial sections DF:dark field; BF: brigth field Fig.
  • AA tela choroidea of third ventricule, thalamus (pulvinar); Bl', antirior hom of the lateral ventricule; CC, co ⁇ us callosum,tela chorioidea of third ventricule, thalamus; DD', Fourth ventricle and cerebellar vermis; EE', genu of the internal capsule; GG', cerebellar peduncle, ependimal channel, pyramis.
  • AA' thalamus, internal capsule, posterior thalamic radiation.
  • BB' white anterior commissure, co ⁇ us callosum
  • DD' putamen, nucleo caudato, anterior arm internal capsule
  • EE' third ventricle, globus pallidum, internal capsule
  • FF' mid brain.
  • GG' hippocampus, controlateral hemisphere
  • HH' fimbria, controlateral hemisphere.
  • A spinal cord sagittal section
  • BB', CC, DD', EE' representative spinal cord of the encephalic- thoracic spinal cord section: BB', C3 section, CC, CX section, DD' T3 section, EE', T8 section
  • Example 10 SK131acZ plasmid construction The following is a scheme for producing the SK131acZ constract. The mutation in the viral UL13 locus creates a new site for the exogenous DNA insertion.
  • HSV-1 fragment Kpnl 17793-K ⁇ nI 28630 was cloned into Kpnl site of ⁇ SP72 plasmid
  • HSV-1 fragment Bglll (then blunt) 25380-KpnI 28630 was cut from pSP72
  • the SK131acZ constract is shown in Figure 46.

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Abstract

La présente invention a trait à un HSV à réplication déficiente codant pour un antigène hétérologue, et à un vaccin comportant le HSV. Le vaccin peut être dirigé contre des virus, des bactéries intracellulaires ou des tumeurs. L'invention a également trait à un vaccin anticancéreux comportant un HSV à réplication déficiente codant pour un inhibiteur de l'angiogenèse, une cytokine et un gène suicide. L'invention a trait en outre à un modèle de provocation pour le criblage d'antigènes candidats, tel qu'un vecteur HSV mutant UL13. L'invention a trait en outre à des vecteurs de HSV pour l'expression de Hex A et des NTF, et à des procédés comprenant ledit HSV.
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WO2009120339A3 (fr) * 2008-03-24 2010-03-18 President And Fellows Of Harvard College Vecteurs pour délivrer des agents neutralisant une maladie
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WO2015123307A1 (fr) * 2014-02-11 2015-08-20 University Of Miami Procédés et compositions pour l'expression d'un transgène dans un système de vecteur du virus de l'herpès
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ES2317749A1 (es) * 2006-07-20 2009-04-16 Consejo Superior Investig. Cientificas Uso de un compuesto proteico derivado de la glicoproteina (gg) de hsv para la elaboracion de una composicion farmaceutica util para la induccion de migracion celular mediada por quimioquinas.
WO2009120339A3 (fr) * 2008-03-24 2010-03-18 President And Fellows Of Harvard College Vecteurs pour délivrer des agents neutralisant une maladie
WO2010039934A1 (fr) * 2008-10-03 2010-04-08 Novartis Ag Compositions du virus de l'herpès bovin-1, vaccin et procédé
US8637046B2 (en) 2008-10-03 2014-01-28 Cornell University Bovine herpes virus-1 compositions, vaccines and methods
WO2014114691A1 (fr) * 2013-01-22 2014-07-31 Vaxxit Srl Utilisation de hsv-1 déficient en réplication en tant que vecteur de vaccin pour la délivrance d'un antigène tat de vih-1
WO2015123307A1 (fr) * 2014-02-11 2015-08-20 University Of Miami Procédés et compositions pour l'expression d'un transgène dans un système de vecteur du virus de l'herpès
US10010594B2 (en) 2014-02-11 2018-07-03 University Of Miami Methods and compositions for transgene expression in a herpesvirus vector system
US11452750B2 (en) 2016-01-27 2022-09-27 Oncorus, Inc. Oncolytic viral vectors and uses thereof
US20200199618A1 (en) * 2016-03-25 2020-06-25 Periphagen, Inc. High-transducing hsv vectors
US10799560B2 (en) * 2016-03-25 2020-10-13 Periphagen, Inc. HSV vectors for delivery of NT3 and treatment of CIPN
US12275949B2 (en) 2016-03-25 2025-04-15 Periphagen, Inc. High-transducing HSV vectors
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