EP1221967A1 - Vaccin a adn cible sur les cellules dendritiques a efficacite elevee - Google Patents

Vaccin a adn cible sur les cellules dendritiques a efficacite elevee

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Publication number
EP1221967A1
EP1221967A1 EP00967758A EP00967758A EP1221967A1 EP 1221967 A1 EP1221967 A1 EP 1221967A1 EP 00967758 A EP00967758 A EP 00967758A EP 00967758 A EP00967758 A EP 00967758A EP 1221967 A1 EP1221967 A1 EP 1221967A1
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European Patent Office
Prior art keywords
vaccine
dna
cd11c
antigen
nucleic acid
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EP00967758A
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German (de)
English (en)
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Thomas Brocker
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Glaxo Group Ltd
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Individual
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Priority to EP00967758A priority Critical patent/EP1221967A1/fr
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—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
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    • 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

Definitions

  • the present invention relates to a vaccine containing a nucleic acid molecule comprising (a) a promoter that is specifically active in antigen presenting cells; and, operatively linked thereto (b) a nucleic acid sequence encoding an antigen.
  • the present invention also relates to a method of vaccinating a mammal wherein the vaccine of the invention is administered to a mammal at a suitable dose.
  • DNA vaccination has been demonstrated to induce protective immunity against experimental cancer and infectious diseases.
  • antigen-encoding DNA is introduced directly into tissues, where the antigen is subsequently synthesized (1).
  • APC antigen presenting cells
  • the present invention relates to a composition containing a nucleic acid molecule comprising (a) a promoter that is specifically active in antigen presenting cells; and, operatively linked thereto (b) a nucleic acid sequence encoding an antigen.
  • said composition is a pharmaceutical composition and most preferably it is a vaccine.
  • antigen presenting cells are professional antigen presenting cells.
  • the promoter must be active in dentritic cells and may be active in macrophages and B cells. Thus, said promoter would not be active, for example, in muscle cells. Promoters useful in accordance with the present invention may be identified by generating differential display libraries for genes from APC. In accordance with the invention, promoters of genes that are predominantly expressed in antigen presenting cells may be employed.
  • Such a promoter is, for example, an MHC class II promoter, a DC-Lamp promoter, a Langerin-promoter or promoters driving expression of cytokines, a promoter driving the expression of an APC specific transcription factor, and their receptors specifically expressed in antigen presenting cells.
  • a promoter driving the expression of an APC specific transcription factor, and their receptors specifically expressed in antigen presenting cells.
  • Such an APC- specific expression can be tested easily by either in vitro transfection of APC or cell lines derived from APC or by in vivo immunisation studies using the modified promoter.
  • operatively linked means, in connection with the present invention, that the nucleic acid encoding said antigen is expressed under the control of said promoter.
  • antigen denotes, in connection with the present invention, a proteinaceous compound that is capable of inducing or triggering an immune response.
  • the vaccine of the present invention is advantageous in several aspects in comparison to prior art DNA vaccines. This holds true for the potency of the immune response as well as the number of immunizations necessary to generate a protective immune response.
  • DC-specific expression of DNA-vaccine antigens is sufficient to induce potent vaccination effects already after one single intramuscular vaccination. This is in clear contrast to conventional DNA-immunizations, where several booster immunizations are necessary to be similarly efficient.
  • the levels of antigen-specific serum titers in mice immunized with the DC-specific construct can be more than one magnitude higher as compared to a conventional DNA-vaccine with ubiquitous expression specificity.
  • a conventional DNA-vaccine construct with ubiquitous expression specificity was compared to an APC-specific DNA vaccine strategy.
  • the latter is exemplified by the DC-specific CD11c-promoter (12-14), while in the conventional DNA vaccine antigen expression was driven by a viral promoter (CMV). It is shown that a DC-specific gene expression results in more efficient immune responses with higher mAb titres as compared to a conventional DNA vaccine.
  • an APC-specific vaccination strategy leads to maximal serum titers of specific Abs already after one single DNA-injection, while the conventional virus promoter driven DNA-construct induces its maximal titres only after a total of three injections.
  • the APC-specific vaccine did not alter the Th-type (Th1/Th2) of the Abs as compared to the conventional DNA-vaccine.
  • the vaccine of the invention can be employed for a variety of purposes.
  • the present invention also relates to the nucleic acid molecule as defined throughout this specification for the preparation of a vaccine for preventing or treating a variety of conditions or for biasing the immune response.
  • the vaccine of the invention can be used to prevent virus infections, bacterial infections or parasite infections. It can further be used to treat or prevent allergies by shifting the TH2 response to a T H 1 response.
  • the vaccine can be used for preventing or treating auto-immune conditions by shifting the T H 1 to a TH2 response or by eliminating the autoimmune T cells.
  • said nucleic acid is DNA.
  • said antigen presenting cells are dendritic cells.
  • said promoter comprises the sequence (aa) TCTCTGCTGGTGCCTCACACGGGCATAC
  • the promoter comprises the sequence of the about 5kb fragment of the mouse CD11c 5'UT described in Example 1.
  • fragments of said promoter optionally also comprising sequences that deviate from said sequences by substitution, deletion, insertion, duplication or inversion and essentially retain promoter function of said sequences are comprised by the present invention.
  • Retainment of the functionality or essentially the functionality of promoter activity can be readily tested by immunizing mice with the modified constructs or using in vitro cell culture assays and transfections with suitable readout systems, such as GFP, Luciferase etc. and checking whether an immune response is generated against the encoded (poly)peptide.
  • suitable readout systems such as GFP, Luciferase etc.
  • the immune response can be assessed quantitatively and qualitatively.
  • An immune response is triggered, if the readout provides a signal at least twice as high as the background level. Appropriate tests are described throughout this specification. However, other suitable tests are also available to the skilled artisan.
  • the "essentially” in connection with retainment of promoter functionality means that at least 70%, preferably at least 80% and more preferably at lest 90% of the functionality of the activity were retained. Activity can be tested as described throughout this specification. The boarder value of at least 70% must be retained in at least one of the activity tests.
  • allergen is well understood in the art and refers to molecules capable of inducing an allergic reaction.
  • said antigen is derived from a pathogen or is derived from an allergen.
  • said pathogen is a bacterium or a virus.
  • said pathogen is an autoantigen involved in auto-immune diseases or a tumor-associated antigen.
  • the nucleic acid molecule may be formulated in said vaccine in a variety of ways, for example in saline.
  • the invention in another preferred embodiment relates to a vaccine wherein said nucleic acid molecule is encapsulated in a cationic liposome or wherein said nucleic acid molecule is coated to a gold particle.
  • the vaccine of the invention may be administered once or several times.
  • said vaccine is a single dose vaccine.
  • single dose vaccine refers to a vaccine that is administered only once and provides a protective immune response. Nevertheless, further vaccinations may further enhance the immune response (e.g. enhance the specific antibody titer or the number of specific T cells).
  • the vaccine comprises a further nucleic acid molecule suitable for expression of a further protein.
  • suitable for expression implies that said further nucleic acid molecule is under the control of a suitable promoter and further necessary regulatory regions that are well-known in the art. These regulatory regions comprise splice sites, transcription start sites, termination signals as well as poly-A attachment sites.
  • Said further protein preferably modifies the immune response, abrogates the immune response (e.g. Fas- ligand, CD30-ligand), members of the TNF family, growth factors).
  • said further protein confers prolongation of survival or enhancement of the function of antigen presenting cells (APCs).
  • Examples of such molecules are bcl-2, TRANCE, cytokines like GM-CSF or chemokines.
  • said APCs are dendritic cells (DC).
  • APC preferably DC-specific expression pattern of the vaccine
  • the molecules of choice exert preferentially a function on the DC (APC) themselves, such as survival-prolongation and enhancement of APC-function (molecule like bcl-2, TRANCE, cytokines like GM-CSF or chemokines).
  • APC a function on the DC
  • they can as well exert functions on other lymphocytes (e.g. T cells) during the process of antigen presentation (like TNF-family members OX40L, CD30L, 4BB1-L, fas-L or costimuiatory molecules).
  • This double expression can be reached by intramuscular coinjection of two plasmids dissolved in saline.
  • One of the plasmids encodes for the antigen, the other for the modifying protein.
  • the APC-specific promoter described above controls the expression of both proteins.
  • both plasmids can be coupled to the same gold-particle in equal amounts and are injected by gene-gun.
  • the invention also relates to a method of vaccinating a mammal comprising administering the vaccine of the invention to a mammal.
  • Vaccination protocols may comprise single or repeated vaccinations.
  • the mammal may be selected from a variety of animals, particularly pets or other domestic animals, said mammal preferably is a human.
  • nucleic acid in general, depends on various factors, which are well-known to the physician in charge. For example, increasing amounts of nucleic acid are required with increasing body weight.
  • Vaccines comprising nucleic acid dissolved in a buffer such as saline would preferably be administered intradermally or subcutaneously. Vaccination via gene-gun technology is preferably done intradermally.
  • the vaccine of the invention is administered as a naked nucleic acid, preferably DNA or as a liposome containing said nucleic acid, preferably DNA, then amounts of 1 yg to 5mg (usually in an aqueous solution, preferably phosphate buffered saline) of active ingredient (i.e. nucleic acid, preferably DNA) is administered per immunization.
  • active ingredient i.e. nucleic acid, preferably DNA
  • amounts of 1 ng to 100 vg of nucleic acid are preferred (see, e.g. BioRad, Genegun Manual).
  • Oral administration is a further option.
  • the amount administered depends, inter alia, on the weight of the patient. In general, all conventional formulation protocols for nucleic acid vaccines can be employed.
  • FIG. 1 The CMV-promoter but not the CD11c-promoter driven construct is expressed in myoblasts. Immunofluorescence analysis of muscle tissue from mice immunized with 50 ⁇ g of different GFP-encoding plasmid constructs. BALB/c-mice were injected with PBS (A, B), CMV-GFP (C, D) or CD11c-GFP (E, F) into the gastrocnemic muscle of their hind legs. 7 days after injection the muscles were surgically excised and histologically analyzed. Photographs were taken at x25 (A, C, E) and x100 (B, D, F).
  • FIG. 2 DC from draining lymph nodes of CMV-GFP as well as CD11c-GFP immunized mice express GFP. Low density cells from draining popliteal lymph nodes were stained with CD11c for detection of DC. Then CD1 1 c + (Fig. 2 CD11 c + ) and
  • CD11c cells (Fig. 2 CD11c ) were further analyzed for expression of GFP in the FL-1 channel of the FACScalibur. Mice were injected either with PBS (Fig. 2a), 100//g of CMV-HA DNA (Fig. 2b) or 100 g of CD11 c-HA DNA (Fig. 2c) into their gastrocnemic muscles 7 days before analysis.
  • FIG. 3 RT-PCR analysis of muscle tissue injected with DNA-vaccines. Muscles from mice vaccinated with 100 g CD11c-HA DNA (Fig. 3a CD11c-HA +), 100 g CMV-HA DNA (Fig. 3a CMV-HA +), mock (PBS) vaccinated muscles from the same animals (Fig. 3a CD11c-HA -, CMV-HA -) were surgically excised and homogenized for extraction of mRNA, which was then transcribed into cDNA with Reverse Transcriptase. To control equal quality of cDNA synthesis, undiluted, 10-fold and 100- fold diluted cDNA was PCR amplified with oligonucieotides specific for HPRT (Fig. 3a).
  • cDNA preparations were compared to amplifications of equal amounts of mRNA before the RT-step (Fig. 3b RT:-).
  • FIG. 4 Immunization with the DC-specific promoter construct elicits stronger and faster antibody responses than the CMV-driven construct.
  • CD11c-plasmid DNA does not contain more immunostimulatory sequences than the CMV-driven construct.
  • Mice were immunized at day 0 and boosted 3 and 6 weeks later in their hind legs as described in the legend to Fig. 1. Three weeks after the last immunization mice were bled at the tail vein. Sera titre were determined by serial dilutions of the non-pooled sera of 5 mice per group by HA-specific ELISA.
  • FIG. 6 Confocal analysis of DC from lymph nodes of CMV-GFP and CD11c-GFP immunized mice.
  • DC were positively selected by CD11c-MACS beads at day 2 (a, b) or day 9 (c, d, e, f) from draining popliteal (a, b, e, f) or non-draining brachial (c, d) lymph nodes of mice immunized into their hind legs with 100mg CMV-GFP (a, c, e) or CD11c-GFP (b, d, f) plasmids.
  • Bright field images were overlaid with the green fluorescence images to show DC and the presence of the vaccination product (GFP). All micrographs were taken at 240x.
  • Figure 7 Coexpression of antigen and OX40L by dendritic cells modifies the antibody response after intramuscular DNA-vaccination.
  • the DNA mixtures used for immunization were either CD11 c-HA plus CD11c-B2M as control plasmid (open bars) or CD11c-HA plus CD11c-OX40L (gray bars) in equal amounts.
  • Figure 8 Gene-gun enforced coexpression of antigen and OX40L by dendritic cells modifies the antibody response after intradermal DNA-vaccination.
  • FIG. 9 Coexpression of antigen and OX40L by dendritic cells after coadministration of CD11c-HA and CD11c-OX40L induces proliferative memory T cell responses.
  • FIG. 10 Coexpression of antigen and OX40L by dendritic cells after coadministration of CD11c-HA and CD11c-OX40L induces memory T cells producing IFN- ⁇ .
  • Supernatants from the T cell proliferation test shown in Figure 9 were collected 4 days after T cell culture. These supernatants were tested for IFN-g content by an IFN-g specific sandwhich-ELISA as described in Materials and methods.
  • mice Mice used in this study were bred in the animal colony of the Basel Institute for Immunology (Basel, Switzerland).
  • MAbs monoclonal antibodies
  • Antisera The mAbs specific for CD19 (No. 09654) and CD11c (No. 09705) were purchased from PharMingen (San Diego, CA).
  • Polyclonal goat anti-mouse IgG and goat anti-mouse IgM sera, as well as subtype specific goat anti-mouse lgG1 and lgG2a were purchased from Southern Biotechnology (Birmingham, AL).
  • HA-specific mAb H36-4-5.2 is described in (15) and was kindly provided by Dr. W. Gerhard (Philadelphia, PA). The corresponding experiments can also be carried out by employing other HA-specific mAbs that are obtainable by conventional procedures.
  • Equivalent cDNAs can be prepared according to conventional procedures; see, e.g. (31 ).
  • the HA-cDNA was cloned as a 1.7kb Xbal blunt-end fragment into the previously described, EcoRI opened, blunt-ended vector pBCD11 c (12). In this vector with 11 kb length, HA-expression is driven by the CD11 c- promoter, while intron and polyadenylation signal are provided by a rabbit ⁇ -globin gene fragment (16).
  • the very same blunt-ended HA cDNA fragment was cloned into the commercially available, EcoRI/Clal opened and blunt-ended vector pBK- CMV(Stratagene, Basel), where cDNA expression is driven by the CMV-promoter and intron sequences as well as polyadenylation signals are derived from SV40.
  • the resulting plasmid had a length of 5.2kb.
  • the integrity of all constructs was controlled by DNA-sequencing. Plasmid DNA was prepared by Qiagen plasmid preparation kit (Qiagen, Hilden).
  • the plasmid pHbAPr-1 -neo-HA was created by cloning the above described blunt end Xbal fragment of HA cDNA into the EcoRI opened and blunt ended vector pHbAPr-1-neo (17).
  • Rolink, Basel which can, for the purposes of this invention, be prepared according to conventional protocols) at 800 HAU/ml in 0.02M NaCI at 4°C for 12h. Plates were washed extensively with PBS and serial threefold dilutions of sera in PBS (phosphate buffered saline), 4% BSA, 0.2% Tween 20 were transferred to the virus coated plates and incubated for 2h at RT (room temperature). After 5 washes with PBS the AP (alkaline phosphatase)- conjugated second step reagent (Goat-anti-mouse IgG, lgG1 or lgG2a, Southern Biotechnology (Birmingham, AL)) was added and incubated for 2h at RT.
  • PBS phosphate buffered saline
  • BSA 4% BSA
  • Tween 20 0.2% Tween 20 were transferred to the virus coated plates and incubated for 2h at RT (room temperature).
  • AP alkaline
  • the AP-substrate p-nitrophenyl phosphate was added according to manufacturer's instructions (Sigma, N-2765) and the coloration quantified at 405 nm with an ELISA-reader (SOFTMAX). Histology. To detect GFP-expression, animals were sacrificed at various time points after vaccine injection and the muscle bundle around the injection site was surgically excised. This tissue was fixed for 12h in PBS, 4% Paraformaldehyde and then embedded in O.C.T. medium (No. 4583; Miles Inc., Elkhart, IN), snap frozen and 20- ⁇ m section were cut with a cryostat for screening of whole muscles.
  • mice Muscle tissue from various mice was isolated and homogenized in 1 ml TriPure Isolation Reagent (Boehringer Mannheim, Germany) per 50mg tissue using a T8.10 Homogenizer (IKA, Staufen, Germany). mRNA was isolated according to manufacturer's instructions. 2 ⁇ g of mRNA was then used for an oligodT-based cDNA synthesis with the SuperscriptTM RNase H " Reverse Transcriptase kit (GibcoBRL, Düsseldorf, Germany).
  • HPRT-specific PCR was performed with graded amounts of cDNA using the HPRT specific oligonucieotides 5'GCTGGTGAAAAGGACCTCT3' and 5'CACAGGACTAGAACACCTGC3'.
  • HPRT specific oligonucieotides 5 ⁇ TGGAATATGTTATCCAGGA3' and 5'GTTTGACACTTCGTGTTACA3' were used.
  • T cell proliferation assay In vitro T cell proliferation assay. After immunisation draining and non-draining lymph nodes were isolated and teased through a steel mesh in order to receive single cell suspensions. These cells were resuspended in T cell culture medium (IMDM 10% FCS (GIBCO, Grand island, NY)) and cultured at adensity of 5x10 5 cells/200 ⁇ l for 5 days in presence or absence of antigen hemagglutinin (HA). Proliferation was measured after addtion of 3 H-Thymidin for the last 8h of culture.
  • HA-cDNA expression was driven by the CMV immediate early promoter with splice- and poly(A) sites derived from SV40.
  • This expression system for eukaryotic cDNA is commercially available with minor modifications depending on the source (pCI, Promega; pCMV, Clontech; pcDNA, Invitrogen; pBK-CMV, Stratagene) and is the most widely used for DNA-vaccination studies. Strong viral promoters with broad cell type specificity such as CMV (and RSV) promoters have been described to generate the most consistently high levels of gene expression (18).
  • the CMV/SV40 system has been successfully used as a DNA-vaccine for the HA antigen (11 ). It was further described to successfully induce immune responses against other antigens (Ag), when the corresponding cDNAs were introduced into the expression cassette (4); (19); (20).
  • Ag antigens
  • This promoter was initially tested by a transgenic approach with random integration of various CD11 c-constructs into the genome of transgenic mice. So far mice derived from more than 15 individual transgenic founders have been tested for expression of various transgenes. All animals showed transgene expression exclusively in lymphoid and myeloid DC of all organs, but not on other cell types (12, 14), underlining the stringent regulation by the DC-specific CD11c-promoter.
  • GFP green fluorescent protein
  • GFP-expression can clearly be detected in muscle fibres of CMV-GFP injected muscle bundles (Fig.l C, D), while CD11c-GFP injection did not lead to detectable expression of GFP (Fig.l E, F).
  • the background fluorescence visible even in the PBS injected muscle in Fig.l A, B is due to autofluorescence induced by the thickness (20 ⁇ m) of the sections. In no case at no timepoint was it possible to detect any green fluorescence above background in muscle tissue injected with CD11c-GFP.
  • CD11c-GFP construct In order to investigate if the CD11c-GFP construct would be functional, DC from the draining popliteal lymph nodes was analyzed for the presence of GFP-expressing DC. Similar numbers of CD11 c + GFP + DC were found in lymph nodes draining CMV-GFP (Fig. 2b) as well as CD11 c-GFP injected muscles (Fig. 2c). In both cases mainly CD11c positive cells expressed GFP (Fig.2, CD11 c + ), while no substantial numbers of GFP-expressing cells were found in the CD11 c-negative fraction (Fig.2, CD11c " ) . This indicates that in contrast to the CMV-driven vaccine, which is expressed in both, muscle cells (Fig. 1 ) and DC (Fig. 2), the CD11c-driven construct is expressed only in DC (Fig. 2), but not in muscle fibres (Fig. 1 ).
  • RNA extracts from injected as well as uninjected muscle bundles 100 ⁇ g of CMV-HA or CD11 c-HA constructs were injected into the gastrocnemic muscle of the hind legs of BALB/c mice and the muscle bundles were removed 5 days post injection.
  • RNA isolation a reverse transcriptase (RT) reaction was performed and in a PCR with oligonucieotides specific for HPRT the presence of similar amounts of cDNA in all samples (Fig. 3a) was controlled. The PCR reaction was then repeated with oligonucieotides specific for HA to determine whether the injected vaccines were expressed in the muscle tissues. As shown in Fig.
  • Example 2 CD11c-promoter Is More Efficient in Inducing Ab-responses than the CMV-promoter Driven Construct.
  • mice were immunized with various amounts of the two different HA-encoding vaccines intramuscularly and bled regularly in order to determine the serum titre of HA-specific Abs. Each animal was bled one day before each immunization and three weeks after the third injection. Vaccines were injected in three week intervals at week 0, 3 and 6. The titres of HA-specific Abs showed a significant increase in all immunized animals of each group (Fig. 4). A comparison of kinetics and strengths of the anti-HA responses in the different experimental groups indicates a clear superiority of the CD11c-promoter driven HA-cDNA.
  • the HA-specific Ab-titre induced by CD11cHA-DNA was approximately 30-fold stronger than the responses induced by the CMV-driven HA- construct after one single injection (Fig. 4a). After the second and third immunization this difference decreased to 23-fold and 14-fold, respectively. This indicates that the kinetics of the responses were clearly different. While the CMV-HA construct induced an increase of anti-HA titre after repeated DNA injections only, the DC-specific construct reached the maximal response already after the first initial injection. In the groups of mice receiving higher doses of CD11c-HA DNA-vaccines, the relative kinetics of the anti-HA titre was similar (Fig. 4b).
  • Example 3 Higher Efficiency of CD11c-promoter Is Not Due to CpG- methylation.
  • mice were immunized with 20 ⁇ g CMV-HA DNA and the serum antibody titres were compared to those of mice immunized with a 1 :1 mixture of 20 ⁇ g CMV-HA and 20 ⁇ g pBCD11c (Fig. 5).
  • the latter construct contained all sequences of CD11c- HA except HA-encoding cDNA (see Materials and Methods). Both immunizations yielded similar levels of HA-serum titres indicating that the CD11 c-construct does not contain additional CpG motives being responsible for its higher stimulatory capacities : (Fig. 5).
  • Example 4 The Different Constructs Generate Ab-responses with Comparable Isotypes.
  • CMV-HA injections induced a greater degree of isotype skewing as indicated by smaller lgG1/lgG2a ratios than CD11c-HA immunization.
  • Booster DNA immunizations altered the quantity of anti-HA Ab, but had no influence on the isotypic nature of the response.
  • our results indicate that a DC-specific expression of DNA vaccine antigen increases the potency of the vaccine as compared to conventional DNA vaccines, but had no influence on the isotypic nature of the response.
  • DC do express antigen encoded by the different vaccines
  • CMV-GFP or CD11c-GFP plasmids and isolated DC at various time points (day 2, 6, 9 post injection; Fig.6 and data not shown) from draining lymph nodes (popliteal lymph nodes).
  • DC should be transfected by the vaccines and therefore express the encoded GFP.
  • Isolated CD11c + DC were therefore analyzed for GFP-expression by confocal microscopy as shown in Fig. 6. Already 2 days post immunization a strong green-fluorescence could be detected in DC from CD11 c-GFP immunized mice.
  • Example 6 Modification of the immune response by DC-specific coexpression of Antigen and OX40L after intramuscular administration of DNA-vaccines.
  • OX40L a member of the TNF family, believed to shift immune responses towards a TH2 type (35, 36).
  • the cDNA encoding OX40L was cloned into the expression cassette of the CD11c-vector described above. This construct was mixed with the CD11c-HA plasmid at a ratio 1 :1 and 2x100 ⁇ g of this mixture were injected i.m. into the hind legs of BALB/c mice ( Figure 7). As a control, the second group of mice received the same CD11c-HA vector mixed with CD11c-B2M.
  • the latter vector encodes beta-2-Microglobulin (B2M), a molecule which does not affect the immune response (the mixture CD11c-HA + CD11c-32M induces the same immune response as CD11c-HA alone).
  • B2M beta-2-Microglobulin
  • Mice were immunized 3 times in 3 week periods and the sera were analysed as described before for HA-specific Ab-responses.
  • the control group ( Figure 7, CD11c-HA + CD11c-B2M, open bars) mounted an HA-specific antibody response of a Th1-type, dominated by lgG2a antibodies.
  • Example 7 Modification of the immune response by DC-specific coexpression of Antigen and OX40L after intradermal administration of DNA-vaccines by the gene gun method.
  • two groups of mice were immunized with the gene-gun method.
  • Gold-particles used for this method were coated either with CD11c-HA plus CD11c-OX40L (Fig.8 A, C, E) or CD11 c-HA plus a control vector (CD11c-B2M) (Fig.8 B, D, F) in a ratio of 1 :1 each.
  • CD11c-B2M CD11 c-B2M
  • Table 1 Isotype analysis of sera from DNA-vaccinated mice.
  • BALB/c mice were immunized three times in three-weeks intervals with the indicated amounts of DNA either in saline or in a liposomal formulation.
  • Blood was taken from the tail veins at weeks 3 and 9 and analyzed for its HA-titre by ELISA as described for Fig. 1.
  • HA- specific antibodies in the non-pooled sera of 5 to 6 mice per group were detected with antiserum specific for either lgG1 or lgG2a.
  • Dendritic Cells Can Induce Negative but not Positive Selection of Thymocytes in vivo. J Exp Med 185:541 -550.

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Abstract

La présente invention concerne un vaccin contenant une molécule d'acide nucléique comprenant: (a) un promoteur spécifiquement actif dans les cellules présentatrices d'antigène; et, fonctionnellement reliée au promoteur précité, (b) une séquence nucléotidique codant un antigène. L'invention se rapporte également à un procédé de vaccination d'un mammifère selon lequel on administre à un mammifère une dose adéquate du vaccin de l'invention.
EP00967758A 1999-09-23 2000-09-22 Vaccin a adn cible sur les cellules dendritiques a efficacite elevee Withdrawn EP1221967A1 (fr)

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