EP1504107A1 - Virus de variole aviaire recombinant - Google Patents

Virus de variole aviaire recombinant

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Publication number
EP1504107A1
EP1504107A1 EP03722604A EP03722604A EP1504107A1 EP 1504107 A1 EP1504107 A1 EP 1504107A1 EP 03722604 A EP03722604 A EP 03722604A EP 03722604 A EP03722604 A EP 03722604A EP 1504107 A1 EP1504107 A1 EP 1504107A1
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EP
European Patent Office
Prior art keywords
recombinant
virus
viruses
gene
fwpv
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EP03722604A
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German (de)
English (en)
Inventor
Robert Baier
Denise Cancer Research UK Oncology Unit BOULANGER
Volker Erlfe
Gerd Sutter
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Publication of EP1504107A1 publication Critical patent/EP1504107A1/fr
Withdrawn legal-status Critical Current

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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a recombinant fowlpox virus (FWPN) and a D ⁇ A nector, which contains gene sequences for such a recombinant fowlpox virus.
  • the invention further relates to a pharmaceutical composition which comprises the recombinant fowlpox virus or a D ⁇ A nector, the use of the recombinant fowlpox virus for the treatment of infectious diseases or tumor diseases and a method for producing the recombinant fowlpox virus or D ⁇ A vector ,
  • the present invention relates to eukaryotic cells or prokaryotic cells which contain the recombinant D ⁇ A nector or the recombinant fowlpox virus.
  • Smallpox viruses of various types have already been established as recombinant vaccine vectors (Moss, 1996; Paoletti, 1996).
  • Bird pox viruses including fowlpox viruses (Fowlpox-Niren or FWPN) as a prototypical member, are known to replicate only in bird cells. In mammalian cells, virus replication is blocked at different times in the replication cycle depending on the cell type, but there is virus-specific gene expression (Taylor et al., 1988; Somogyi et al., 1993).
  • Shotgun insertion strategies have been used to identify several insertion sites in the FWPV genome (Taylor et al., 1988; Jenkins et al., 1991).
  • the first strategy is the frequently used transient dominant selection method described by Falkner and Moss (1990), in which the selection marker is present in the plasmid sequence outside the insertion cassette.
  • Recombinant viruses which are generated by a simple crossover event and which contain the entire plasmid sequence are obtained in the presence of selection medium. These recombinant viruses are unstable due to the presence of the repeated sequences of the flanking regions.
  • the marker gene lying between these repeats is deleted after a second recombination, which either leads to the production of the wild-type (wt) virus or a stable recombinant virus. The latter must be isolated again after the plaque procedure and then identified by PCR or Southern blotting.
  • a second method is based on the observation that in the case of recombinant FWPV which expressed the target protein and ⁇ -galactosidase under the control of the P7.5 promoter in a directly repeated orientation, a homologous recombination took place between the promoter repeats, whereby the lacZ gene is deleted (Spehner et al., 1990). Therefore, white plaques were formed from recombinant viruses that had lost the marker gene.
  • a similar strategy has been developed to produce recombinant MVA viruses using the regulatory vaccinia virus KIL gene as a transient selection marker, which is removed by means of intragenomic homologous recombination (Staib et al., 2000).
  • FWPV grows more slowly than the vaccinia virus. Maintaining the full replication ability of recombinant viruses is critical to the generation and use of potential FWPV vaccine viruses.
  • the present invention is based on the tasks of providing a recombinant fowlpox virus which results in increased vector stability after insertion of foreign DNA and a higher level of safety when used as a vaccine vector, while maintaining full replication capability and optimal efficiency during the Selection of recombinant viruses retained.
  • the solution according to the invention is based on the identification of the FWPV-Fl III gene as a new insertion site for foreign DNA.
  • F1 IL mutated viruses replicated efficiently after infection with CEF (chicken embryo fibroblasts).
  • CEF chicken embryo fibroblasts
  • the F III of the FWPV is already known and precisely identified. In the publication by Afonso et al. In 2000, the Fl III gene homolog was precisely identified as ORF FPV110 with genome position 131.387-132.739. Afonso et al. however, do not disclose the property of the Fl IL gene as an integration site for foreign DNA.
  • Fl IL gene as an integration site for foreign DNA offers some unexpected advantages: First, it has surprisingly been found that the recombinant fowlpox viruses, which contain one or more insertions of foreign DNA in the Fl III gene, are one over conventional ones Vectors have increased vector stability. In addition, the recombinant FWPV according to the invention have proven to be very safe in the in vivo application as vaccine ectors. Another advantage of the insertion according to the invention into the F1 IL gene can be seen in the fact that the insertion can be carried out at any point in the gene.
  • the present invention consequently provides a recombinant fowlpox virus (FWPV) which contains at least one insertion of a foreign DNA in the Fl 1L gene.
  • the insertion takes place in position 131.387-132.739 of the FWPV genome.
  • the insertion can in principle take place at any desired location of the Fl IL gene, an insertion in the genome section defined by the nucleotide positions 131.860-131.870 in the fowlpox virus genome is preferred.
  • Any DNA that is introduced into the DNA of an organism, a cell or a virus etc. using genetic engineering methods is generally referred to as foreign DNA in the context of the present invention, from which it does not originate.
  • the foreign DNA contains at least one foreign gene, optionally in combination with a sequence for regulating the expression of the foreign gene.
  • the foreign gene contained in the recombinant fowlpox virus (FWPV) according to the invention codes for a polypeptide which can preferably be used therapeutically and / or codes for a detectable marker and / or is a selection gene.
  • a reporter gene refers to genes whose gene products can be detected using simple biochemical or histochemical methods. Indicator and marker genes are synonyms for the term reporter gene.
  • a selection gene or selection marker denotes genes which give viruses or cells in which the corresponding gene products are formed a growth advantage or survival advantage over other viruses or cells which do not synthesize the corresponding gene product.
  • Selection markers preferably used are the genes for E. coli guanine phosphoribosyl transferase, E. coli hygromycin resistance and neomycin resistance.
  • the foreign DNA sequence can be a gene which codes, for example, for a pathogenic agent or for a component of a pathogenic agent.
  • Pathogenic agents are understood to mean viruses, bacteria and parasites that can cause an illness, as well as tumor cells that multiply uncontrollably in an organism and can therefore lead to pathological growth. Examples of such pathogenic agents are described in Davis, BD et al., (Microbiology, 3rd edition, Harper International Edition).
  • Preferred pathogenic agents are components of influenza viruses or measles and of respiratory syncytial viruses, of dengue viruses, of human immunodeficiency viruses, for example HTV I and HIN II, of human hepatitis viruses, for example HCN and HBN, of herpes -Niren, Papilloma-Niren, the malaria parasite Plasmodium falciparum and the tuberculosis-causing mycobacteria.
  • components of pathogenic agents are, for example, envelope proteins of viruses (HTV-Env, HCV-E1 / E2, influenza virus-HA- ⁇ A, RSV-FG), regulatory virus proteins (HTV-Tat-Rev- ⁇ ef, HCV- ⁇ S3- ⁇ S4- ⁇ S5), the protective antigen protein from Bacillus anthracis, Merozoite Surface Antigen and circumsporozoite protein from Plasmodium fal- ciparum, the tyrosinase protein as a melanoma antigen, or the HER-2 / neu protein as an antigen of human adenocarcinomas.
  • envelope proteins of viruses HTV-Env, HCV-E1 / E2, influenza virus-HA- ⁇ A, RSV-FG
  • regulatory virus proteins HTV-Tat-Rev- ⁇ ef, HCV- ⁇ S3- ⁇ S4- ⁇ S5
  • the protective antigen protein from Bacillus anthracis
  • Preferred genes which code for tumor-associated antigens are those for melanoma-associated differentiation antigens, for example tyrosinase, tyrosinase-related proteins 1 and 2, from cancer testes antigens or tumor testicular antigens, for example MAGE 1, -2, -3 and BAGE, for non-mutated shared antigens or antigens which are shared by several tumor types which are overexpressed on tumors, for example Her-2 / neu, MUC-1 and p53, are encoded.
  • melanoma-associated differentiation antigens for example tyrosinase, tyrosinase-related proteins 1 and 2
  • cancer testes antigens or tumor testicular antigens for example MAGE 1, -2, -3 and BAGE
  • non-mutated shared antigens or antigens which are shared by several tumor types which are overexpressed on tumors for example Her-2 / neu, MUC-1 and p53
  • Polypeptides which are a component of HTV, Mycobacterium spp. are particularly suitable. or Plasmodium falciparum or are part of a melanoma cell.
  • constituents are to be understood as constituents of the aforementioned which have immunogenic properties, that is to say are able to produce an immune reaction in mammals, in particular humans (for example surface antigens).
  • promoters are known to the person skilled in the art, for example a pox virus-specific promoter can be used.
  • the detectable marker is a beta-galactosidase, beta-glucoronidase, a luciferase or a green fluorescent protein.
  • the marker gene and / or selection gene can be eliminated.
  • this property is of great advantage because it enables the same selection strategy for the insertion to be repeated at different points.
  • the presence of a marker gene is also not recommended for a vaccine for human use.
  • the deletion of these gene sequences The genome of the final recombinant viruses is virtually "automatically" by an intragenomic homologous recombination between identical gene sequences that flank the marker selection gene expression cassette.
  • the present invention provides a DNA vector which contains a recombinant fowlpox virus according to the invention or functional parts thereof which contain at least one insertion of a foreign DNA in the Fl III gene, and further preferably a replicon for replicating the vector in a pro- or eukaryotic cell and a selection gene or marker gene which can be selected in pro- or eukaryotic cells.
  • a recombinant fowlpox virus according to the invention or functional parts thereof which contain at least one insertion of a foreign DNA in the Fl III gene, and further preferably a replicon for replicating the vector in a pro- or eukaryotic cell and a selection gene or marker gene which can be selected in pro- or eukaryotic cells.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook, et al, in Molecular Clo ning. A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, described New
  • the DNA vectors of the present invention play the role of a self-contained, replicable entity that possesses the ability to replicate DNA in suitable host cells.
  • the non-replicable foreign DNA is thus passively replicated and can then be isolated and purified together with the vector.
  • the DNA vector can also contain the following sequence elements: enhancers for enhancing gene expression, promoters which are the prerequisite for gene expression, origins of replication, reporter genes, selectable genes, splice signals and packaging signals.
  • the DNA vector according to the invention serves primarily as a transfer vector in order to enable the insertion of foreign genes in a virus-infected cell via homologous recombination. It is usually used in the context of a Fowlpox virus infection, since the regulatory elements depend on the presence of other virus proteins.
  • the recombinant fowlpox virus or the DNA vector is provided in a pharmaceutical composition, which these in combination with pharmaceutical includes compatible excipients and / or carriers.
  • the pharmaceutical composition is preferably a vaccine.
  • the FWPV generated according to the present invention are converted to a physiologically acceptable form. This can be done on the basis of many years of experience in the preparation of vaccines which are used for vaccination against smallpox (Kaplan, Br. Med. Bull. 25, 131-135 [1969]).
  • PBS phosphate buffered saline
  • peptone and 1% human albumin in an ampoule, freeze-dried, preferably in a glass ampoule.
  • the lyophilisate can contain fillers or diluents (such as, for example, nitro nol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other auxiliaries (for example antioxidants, stabilizers, etc.) which are suitable for parenteral administration.
  • fillers or diluents such as, for example, nitro nol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone
  • auxiliaries for example antioxidants, stabilizers, etc.
  • the lyophilisate can be dissolved in 0.1 to 0.2 ml of an aqueous solution, preferably physiological saline, and administered parenterally, for example by intradermal inoculation.
  • the vaccine according to the invention is preferably injected intracutaneously. Slight swelling and redness, and sometimes itching, may occur at the injection site.
  • the route of administration, the dose and the number of administrations can be optimized in a known manner by the person skilled in the art. Where appropriate, it is convenient to administer the vaccine several times over an extended period of time to achieve a high level of immune responses to the foreign antigen.
  • the aforementioned objects according to the invention ie the recombinant fowlpox virus, the DNA vector or the pharmaceutical composition are preferably used for the treatment of infectious diseases or tumor diseases as defined above.
  • the Fowlpox virus according to the invention, the DNA vector or the pharmaceutical composition can be used prophylactically or therapeutically either alone (for example as a vaccine) or in the context of a so-called prime boost approach.
  • the immune response against the fowlpoxvirus vaccine can be increased further by repeated administration of a vaccine dose of the fowlpoxvirus according to the invention.
  • Fowlpox viruses according to the invention with other viral vectors, for example MVA.
  • MVA or other vaccine viruses that belong to the genus of orthopoxviruses can be used.
  • certain strains of the vaccinia virus have been used as live vaccine for smallpox immunization for many years, for example the Elstree strain of the Lister Institute in the United Kingdom.
  • Vaccinia viruses have also been used frequently as vectors for the generation and delivery of foreign antigens (Smith et al., Biotechnology and Genetic Engineering Reviews 2, 383-407 [1984]).
  • Vaccinia viruses are among the best studied living vectors and have special features that support their use as recombinant vaccines: they are highly stable, inexpensive to manufacture, easy to administer and can absorb large amounts of foreign DNA.
  • the vaccinia viruses have the advantage of inducing both antibody and cytotoxic reactions and enable the presentation of antigens to the immune system in a more natural way and have been used successfully as vector vaccines that protect against infectious diseases.
  • vaccine viruses are infectious to humans and their use as an expression vector in the laboratory is restricted by safety concerns and regulations.
  • Most of the recombinant vaccinia viruses described in the literature are based on the Western Reserve (WR) strain of the vaccinia viruses.
  • WR Western Reserve
  • this strain is highly neurovirolent and therefore poorly suited for use in humans and animals (Morita et al, Vaccine 5, 65-70 (1987)).
  • Safety concerns regarding the standard strains of W have been addressed through the development of vaccine vectors from highly attenuated virus strains, which are characterized by their limited ability to reproduce in vitro and their avirulence in vivo.
  • the so-called modified vaccine virus Ankara (MVA) was cultivated based on the Ankara strain.
  • the MVA virus was deposited with CNCM on December 15, 1987 under the deposit number I-721 in accordance with the requirements of the Budapest Treaty.
  • vaccinia viruses and poxvirus vectors with similar properties can also be used for the above vaccination schedule, e.g. recombinant forms of the vaccinia viruses NYVAC, CN-I-78, LC16m0 and LC16m8 as well as recombinant parapox viruses such as e.g. the attenuated Orf virus D1701.
  • adenoviruses in particular human adenovirus 5
  • orthomyxoviruses in particular influenza viruses
  • herpes viruses in particular human or equine herpesviruses
  • alphaviruses in particular Semiliki-Forest-Niren, Sindbisviruses, and equine encephalitis
  • the Fowlpox detector according to the invention is preferably used during the first vaccination, i.e. during priming.
  • a vaccination scheme according to the invention which can be used, for example, as part of a vaccination against infectious diseases or tumor diseases, or for the treatment thereof, is as follows:
  • a method according to the invention for immunizing an animal preferably a human, preferably comprises the following steps:
  • the priming step is preferably carried out twice before the boosting step and particularly preferably the priming steps are carried out at the start of the treatment and in weeks three to five, preferably week four, of the immunization, the boosting step being carried out in weeks eleven to thirteen, preferably week twelve of immunization is carried out.
  • the present invention is also directed to a combined preparation for the successive use of the individual components mentioned above, for vaccination.
  • a combined preparation consists of the following components:
  • Prime / Boost regulation provides a better immune response than vaccination with either Fowlpox viruses according to the present invention or another vector such as MVA alone.
  • the method according to the invention for producing a recombinant fowlpox virus or DNA vector comprises introducing foreign DNA into the F1 III gene of a fowlpox virus by means of recombinant DNA techniques.
  • the introduction is preferably carried out by homologous recombination of the virus DNA with the foreign DNA, the Fl 1L- contains specific sequences, followed by replication and isolation of the recombinant virus or the DNA vector.
  • the present invention further provides eukaryotic cells or prokaryotic cells which contain the recombinant DNA vector according to the invention or the recombinant FWPV.
  • a bacterial cell preferably E. coli cell
  • Avian cells preferably chicken cells, or a mammalian cell, preferably a human cell, can be used as eukaryotic cells, with the exception of human embryonic stem cells and human germline cells.
  • the DNA vector according to the invention can be transfected into the cells, for example by means of calcium phosphate precipitation (Graham et al., Virol. 52, 456-467 [1973]; Wigler et al., Cell 777-785 [1979] by means of electroporation (Neumann et al., EMBO J. 1, 841-845 [1982]), by microinjection (Graessmann et al., Meth. Enzymology 101, 482-492 (1983)), by means of liposomes (Straubinger et al., Methods in Enzymology 101, 512-527 (1983)), by means of spheroblasts (Schaffner, Proc. Natl. Acad. Sci. USA 77, 2163-2167 (1980)) or by other methods known to the person skilled in the art Transfection using a calcium phosphate precipitation is preferably used.
  • calcium phosphate precipitation Graham et al., Virol. 52, 45
  • Fig. 1 (A) Primer walking sequencing strategy for the sequencing of FWPV-Fl 1L. The length of each sequence reaction is shown. (B) Schematic representation of the FWPV genome, showing the inverted terminal repeats (ITR) and the central location of the Fl IL gene, as well as a representation of the production of Fl III gene sequences which were used as flanking sequences for the homologous recombination , It is the positions along the F1 IL ORF for the primers F1 and F2 that are used to amplify Flank 1 was used, as well as primers F3 and F4 used to amplify Flank 2.
  • ITR inverted terminal repeats
  • a unique PweER restriction site in pLGFV7.5 can be used to insert foreign genes that are placed under the transcriptional control of P7.5.
  • the mouse tyrosinase-encoding gene (mTyr) served as the first recombinant model gene.
  • Fig. 4 Multi-step growth curve experiment. CEF were inoculated in triplicate with either FP9 virus or with the FL III knockout virus in a moi of 0.05 pfu / cell. The triple batches were harvested at different times after infection and titrated under agar. The error bars show the standard deviations between the triple samples. 5: PCR analysis of genomic DNA from the recombinant FWPV tyrosinase virus MT31. The initial plaque isolation (lane 0) and the first 2 subsequent plaque cleaning rounds (lanes 1 and 2) took place in the presence of selection medium (MXH), whereas the last 3 plaque cleaning steps (lanes 3 to 6) took place in the absence of MXH.
  • MXH selection medium
  • pLGFV7.5-mTyr-DNA was used as the control template DNA
  • FP9 is the wt virus control DNA
  • UC the non-infected control DNA.
  • Control PCR (F1-F2) showing the relative amount of virus DNA.
  • PCR F1-F4 the 984 bp band corresponds to the expected DNA fragment which is amplified from wt virus DNA (wt), the 7282 bp band corresponds to the amplification product which contains the tyrosinase gene and the / Contains ⁇ cZ-gpt sub-cassette which are contained in the intermediate recombinant virus (interm.), the 2880 bp band corresponds to the product which is only the amplification product of the tyrosinase gene expression cassette (rec.).
  • C PCR PR43-44 showing the presence of the / ccZ sequence.
  • D Expression of mouse tyrosinase, which is detected by the production of melanin in CEF.
  • CEF cells in 6 cm diameter petri dishes were infected with a moi of 0.1 pfu / cell. Six days after infection, the cells were harvested, transferred to a U-bottom microtiter plate and washed in PBS. Lanes 1-5: cells infected with five different recombinant viruses; Lane 6: uninfected cells; Lane 7. cells infected with wt virus.
  • Fig. 6 Advantage of a combined vaccination with FWPV tyrosinase and MVA tyrosinase vaccines in the prime boost method. Two mice per group were immunized twice every four weeks with 10 8 infectious units of virus vaccine by intraperitoneal administration.
  • the vaccination groups are made up as follows:
  • the T cells from the spleens of the animals were prepared, cultivated in vitro over a period of 7 days and then tested for their cytotoxic capacity for tyrosinase-specific target cells in the chromium release test. This shows the values obtained for the specific lysis of the target cells (in% with an effector / target ratio of 30: 1). It was shown that the T cells of the combined vaccinated animals clearly had the highest reactivity in the FM group. In contrast, only moderate cytotoxic responses could be measured in the FF and MM group mice immunized uniformly with regard to the vaccine. The weakest cytotoxicity was shown in the test of the group MF T cells, which had been vaccinated first with MVA tyrosinase and then with FWPV tyrosinase.
  • CEF Primary chicken embryo fibroblasts
  • MEM MEM
  • BMS basal medium supplement
  • HeLa cells and Vero cells were grown in DMEM (Gibco) enriched with 10% fetal calf serum (FCS) (Gibco).
  • FCS fetal calf serum
  • FWPV-FP9 a well characterized plaque isolate of the attenuated strain HP1-438 (Boulanger et al., 1998), was grown for CEF in the presence of MEM enriched with 2% FCS.
  • FWPV-FP9 grown on CEF were harvested after a freeze-thaw cycle.
  • the virus was concentrated by ultracentrifugation and semi-purified through a 25% (w / w) sucrose cushion as previously described (Boulanger et al., 1998).
  • the sediment was resuspended in 0.05 M Tris, pH 8, with 1% SDS, 100 ⁇ M ⁇ -mercaptoethanol and 500 ⁇ g / ml Proteinase K and incubated for 1 hour at 50 ° C. After phenol / chloroform extraction, the DNA was isolated, precipitated with ethanol and resuspended in H 2 O. Sequencing was carried out using primer walking on the virus DNA.
  • the first primer (PR30) was designed based on the partial sequence of the pigeonpox Fl IL gene, which was developed by Ogawa et al. (1993) was published under accession number M88588.
  • the primers used for sequencing were: PR30: 5'-CTCGTACCTTTAGTCGGATG-3 ⁇ PR31: 5'-GGTAGCTTTGATTACATAGCCG-3 ', PR32: 5'- GATGGTCGTCTGTTATCGACTC-3' and PR33: 5'- GTCTGATAGATGTAAAT.
  • a 4.2 bp / ⁇ cZ-gpt cassette which corresponds to the cassette contained in the plasmid p ⁇ iLZgpt described by Sutter & Moss (1992) and the E. coli lacZ gene under the control of the late vaccinia virus promoter Pl 1 and the E. coli gpt gene under the control of the early / late vaccinia virus promoter P7.5 was inserted directly into the multiple cloning site of the pBluescript II SK + plasmid (Stratagene), plasmid pBSLG was obtained.
  • GGCCGCGGCCGCCACTAGATGAACATGACACCGG-3 ' GGCCGCGCGCCACTAGATGAACATGACACCGG-3 '
  • PRF2 5'- GGCCCCCCGGGGCATTACGTGTTGTTTGTTGC-3'
  • This fragment was used in pBSLG previously digested with the same enzymes, pBSLGFl 1 being obtained.
  • Flank 2 (534 bp) was primed using the primers PRF3 (5'-GGCCCCTGCAGGCAACAAACAACACGTAATGC-3 ') and PRF4 (5'-CGCCCGTCGACCTTCTTTAGAGGAAATCGCTGC-3'), which contain a Pstl and S ⁇ / 7 restriction site , This fragment was inserted into pBSLGFl 1 which had previously been digested with both enzymes, pLGFl 1 being obtained.
  • the Flank 2 repeat P7.5 promoter cassette was then extracted from the plasmid obtained by RstZ cleavage. cut, treated with Klenow polymerase and inserted into the Sm ⁇ J site of pLGFl 1, the insertion plasmid pLGFV7.5 being obtained.
  • pLGFV7.5-rnTyr A single E ⁇ ne / site downstream of the vaccinia virus P7.5 promoter sequence in plasmid pLGFV7.5 was used to insert the gene coding for mouse tyrosinase into this plasmid.
  • the plasmid pZeoSV2 + / muTy (Drexler et al., Unpublished results) was cleaved with Nhel and Not /. The desired fragment was treated with Klenow polymerase and inserted into the Pmel restriction site with blunt ends in pLGFV7.5, whereby the plasmid pLGFV7.5-mTyr was obtained.
  • CEF infected with FWPV FP9 were transfected with plasmid pLGFl 1 using Lipofectin (Gibco).
  • the progeny virus was harvested and plated under agar containing mycophenolic acid, xanthine and hypoxanthine (MHX medium).
  • MHX medium mycophenolic acid, xanthine and hypoxanthine
  • Viruses which formed ⁇ -galactosidase positive plaques were visualized with an Xgal coating and the plaques were cleaned twice in the presence of selection medium. LacZ / gpt + viruses were further purified without selection medium until 100% blue plaques were obtained.
  • the total D ⁇ A was isolated from CEF which had been infected with different selected virus isolates, extracted as described above (Boulanger et al., 1998) and analyzed by means of PCR, using the primers PRF1 and PRF4, to check the absence of the wt sequence, and the primers PRF1 and PRF2 to check for the presence of DNA.
  • CEF infected with FWPV FP9 were transfected with linearized pLGFV7.5 mTyr plasmid DNA (Fig. 2).
  • Recombinant viruses were purified three times in the presence of selection medium.
  • blue plaque isolates which had once been propagated to CEF, were further purified in the absence of selection medium. Viruses that formed white plaques were then plaque cleaned.
  • the clones obtained were then tested by means of PCR as described above, in addition to which a PCR was carried out using 2 primers specific for the ⁇ cZ sequence (PR43: 5 * -GACTACACAAATCAGCGATTTCC-3 'and PR44: 5'-CTTCTGACCTGCGGTCG-3') , so that the presence of the selection cassette could be examined.
  • the FWPV-Fl III gene is located in the central region of the virus genome (FIG. 1 B). Since the corresponding open reading frame is fragmented in the genome of the CEF-adapted vaccinia virus strain MVA (Antoine et al., 1998), we speculate that the gene may not be essential for FWPV replication.
  • the partial sequence of the C-terminus of the orthopedic Fl IL virus from poxpox virus as well as the complete gene coding for the F12L pox virus virus ortholog and a partial sequence for the F13L ortholog were known (Ogawa et al., 1993; accession number M88588 ).
  • Frame shift mutations of the coding F1 IL sequence in vaccinia virus MVA indicated that F1 1L may be a non-essential gene that may be used as an insertion site.
  • F1 1L may be a non-essential gene that may be used as an insertion site.
  • our analysis of the FWPV-Fl 1L protein (451 amino acids) using the GeneStream Align program showed only 18.6% o amino acid identity with the ortholog (354 amino acids) of the vaccinia virus strain Copenhagen, which indicated different properties in both could indicate viruses.
  • FWPV-Fl 1L In order to determine whether FWPV-Fl 1L can be used as a new insertion site, we constructed mutant viruses using insertion disruption of the coding Fl III sequence.
  • the plasmid pLGFl 1 which contained the / ocZ cassette, flanked by 2 sequences from the FWPV-Fl 1L-ORF (FIGS. 1B and 2), was used to produce recombinant viruses det, which were selected for their growth in the presence of mycophenolic acid under an XGal coating.
  • the recombinants can be obtained from either a double recombination event in both flank 1 and flank 2, resulting in stable recombinant viruses, or by a simple recombination event in one of the flanking gene sequences, which leads to unstable intermediate recombinant genomes. In the latter case, further passages in the absence of selection medium, which make wt virus visible as white plaques, are necessary until a stable recombinant virus is obtained which only produces blue plaques.
  • the genotype of successive virus isolates was characterized by PCR using the external primers used to generate the flanks (PRF1 and PRF4).
  • Virus clone F2 (FIG. 3A) had lost the wt gene sequence after 4 plaque purifications (clone F2.1.2.1.1).
  • Virus clone F 15, which only generated blue plaques after 3 plaque purifications (Fl 5.1.1.1), still contained the wt sequence, as shown by PCR (FIG. 3A).
  • the restricted dilution After amplification of this virus clone (Fl 5.1.1.1.1) by three successive passages in CEF, the restricted dilution also revealed the presence of viruses which produced white plaques.
  • the virus clone F8 only needed a further plaque cleaning in order to be free of wt virus be (Fig. 3B).
  • the plaque titration of virus clone F9.1.1.1.1 after three propagation cycles in CEF showed no presence of contaminating wt virus.
  • F11L as an insertion target enables the stable expression of recombinant genes
  • the plasmid pLGFl 1 was used to construct a plasmid vector (pLGFV7.5) so that foreign genes could be inserted into the FWPV genome together with the / ⁇ cZ-gpt selection sub-cassette under the control of the vaccinia virus P7.5 promoter (FIG. 2).
  • the plasmid also contained a repeat of the Flank-2 sequence (Fig. 2) so that the sub-cassette could then be removed from the recombinant viruses.
  • the first foreign gene obtained from pLGFV7.5 was inserted, the DNA sequence coding for the enzyme tyrosinase, which is of interest as an antigen for an experimental vaccination against melanoma (Drexler et al., 1999).
  • Tyrosinase is involved in the biosynthetic pathway for the production of melanin. Cells that express this enzyme accumulate melanin and go dark. This property provides a simple method for screening for the expression of tyrosinase and its functional integrity. After transfection with pLGFV7.5-mTyr, five recombinant virus clones were selected for further analysis.
  • the linearization of the plasmid DNA which had proven to be very efficient during the production of the F1 IL mutant virus, was also used to produce recombinant viruses.
  • the virus Clones MT22 (data not shown) and MT31 (FIG. 5) showed no detectable wt virus sequence after only one plaque cleaning in the presence of selection medium (MT31.1, lane 1, FIG. 5B).
  • the genomic DNA preparation of both virus clones already showed the presence of recombinant virus genomes which no longer contained any detectable marker gene sequences (2880 bp gene product in FIG.
  • the vaccinia virus Fl 1L ORF potentially encodes a protein that has no homology or characteristic motif that could predict a specific function. Therefore, the FWPV's FIIP ortho may not be essential.
  • this hypothesis was examined by inserting a selection cassette into the FWPV gene which contained a marker gene (lacZ) and a selection gene (gpt).
  • lacZ marker gene
  • gpt selection gene
  • the generation of recombinant viruses containing this cassette and no wt gene sequences showed that the full-length orthologous FWPV gene is not essential for the growth of FWPV.
  • the mutant virus grew just as efficiently as the wt virus (FIG. 4), which suggests that the F1 IL gene locus can be considered a suitable insertion site for recombinant genes.
  • this site to successfully generate recombinant FWPV viruses that stably expressed the melanoma model antigen tyrosinase.
  • the stable expression of marker or selection genes in recombinant viruses can be unsuitable for use as a vector vaccine or for other genetic engineering.
  • the selection sub-cassette was flanked by repeat sequences so that it could subsequently be eliminated.
  • the production of such a recombinant first requires the isolation of a recombinant virus which still contains the selection sub-cassette but no longer contains a wt sequence, and then the isolation of the stable recombinant which has lost the selection sub-cassette.
  • the efficiency of the isolation strategy is therefore critical so that final recombinants can be obtained in a reasonable amount of time.
  • tyrosinase recombinant FWPV viruses obtained by using Fl 1L as a target can easily be monitored by examining the melanin synthesis, simply by examining the color of the cell sediments (Fig. 5D and Table 1).
  • CMV cytomegalovirus
  • Vaccinia virus DNA ligase is nonessential for virus replication - recovery of plasmids from virus-infected cells. Virology 180, 625-
  • Transient host ranks selection for genetic engineering of modified vaccinia virus

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Abstract

L'invention concerne un virus de variole aviaire recombinant (FWPV) et un vecteur ADN contenant des séquences géniques pour un tel virus de variole aviaire recombinant. La présente invention porte également sur une préparation pharmaceutique comprenant ce virus de variole aviaire recombinant ou un vecteur ADN, l'utilisation de ce virus de variole aviaire recombinant pour traiter des maladies associées à des infections ou à des tumeurs, ainsi qu'un procédé pour réaliser ce virus de variole aviaire recombinant ou ce vecteur ADN. Enfin, cette invention concerne des eucaryotes ou des procaryotes comprenant le vecteur ADN recombinant ou le virus de variole aviaire recombinant. Ladite invention est basée sur l'identification des gènes FWPV-F11L en tant que nouvel emplacement d'insertion pour l'ADN étranger.
EP03722604A 2002-05-14 2003-05-13 Virus de variole aviaire recombinant Withdrawn EP1504107A1 (fr)

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CN115300622A (zh) 2015-02-25 2022-11-08 纪念斯隆-凯特琳癌症中心 使用灭活的修饰的痘苗病毒安卡拉作为实体肿瘤的单一免疫疗法或与免疫检查点阻断剂组合
EP3283088A4 (fr) 2015-04-17 2018-10-24 Memorial Sloan-Kettering Cancer Center Utilisation de mva ou de mvadeltae3l en tant qu'agents immunothérapeutiques contre des tumeurs solides
CN105002145B (zh) * 2015-07-01 2017-12-15 天津农学院 利用mTERT和mTyr双启动子联合调控HN基因构建重组腺病毒的方法及重组腺病毒和应用
DE102015111756A1 (de) * 2015-07-20 2017-01-26 Eberhard Karls Universität Tübingen Medizinische Fakultät Rekombinanter Orf-Virus-Vektor
JP7034080B2 (ja) 2016-02-25 2022-03-11 メモリアル スローン ケタリング キャンサー センター ヒトｆｌｔ３ｌを発現する組換えｍｖａまたはｍｖａδｅ３ｌおよび固形腫瘍に対する免疫療法薬としてのそれらの使用
CA3015650A1 (fr) 2016-02-25 2017-08-31 Memorial Sloan Kettering Cancer Center Virus vaccinaux attenues aptes a la replication presentant une deletion de la thymidine kinase avec et sans expression du flt3l ou gm-csf humain pour une immunotherapie anticancereuse
WO2018209315A1 (fr) * 2017-05-12 2018-11-15 Memorial Sloan Kettering Cancer Center Mutants du virus de la vaccine utiles pour l'immunothérapie anticancéreuse

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