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WO1997032987A1 - Procede et compositions nucleiques immunogenes codant pour des antigenes, et molecules co-stimulant l'immunisation - Google Patents

Procede et compositions nucleiques immunogenes codant pour des antigenes, et molecules co-stimulant l'immunisation Download PDF

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Publication number
WO1997032987A1
WO1997032987A1 PCT/CA1997/000162 CA9700162W WO9732987A1 WO 1997032987 A1 WO1997032987 A1 WO 1997032987A1 CA 9700162 W CA9700162 W CA 9700162W WO 9732987 A1 WO9732987 A1 WO 9732987A1
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vector
antigen
host
cytokine
nucleic acid
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PCT/CA1997/000162
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English (en)
Inventor
Brian H. Barber
Neil L. Berinstein
Adrienne K. Chan
Akiko Iwasaki
Niclas B. J. Stiernholm
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University Of Toronto
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Priority to AU18652/97A priority Critical patent/AU1865297A/en
Publication of WO1997032987A1 publication Critical patent/WO1997032987A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • 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
    • 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
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of immunology and is particularly concerned with immunogenic compositions comprising nucleic acid molecules encoding antigens, co-stimulatory molecules and, optionally, cytokines.
  • CTL cytotoxic T-lymphocytes
  • the minimal inflammatory response associated with the injection of plasmid DNA in saline is unlikely to induce the cytokines normally associated with the generation of a strong T-cell response.
  • the key T cell induction events result from the transfection of professional antigen presenting cells (APC) either in the vicinity of or remote from the muscle injection site (ref. 7) .
  • APC professional antigen presenting cells
  • protein products of the transfected gene could be released from muscle cells and mediate T-cell activation as a result of processing by physically remote APCs.
  • CD86 are expressed on activated B-cells and other APCs, and their interactions with either CD28 or CTLA-4 on T-cells provide the obligatory second signal required for MHC restricted T-lymphocyte activation (reviewed in ref. 8) .
  • B7-2 is expressed before B7-1 in a developing immune response and it has been postulated that these two co-stimulatory molecules may have distinct functional features (ref. 9) . In support of this concept, recent studies have shown that the two molecules can have differential effects on promoting specific T-cell effector functions (ref. 10) .
  • IL-12 promotes CTL activity and is a 70 kD heterodimeric cytokine comprised of p35 and p40. It is most commonly produced by cells of the macrophage/monocyte lineage (reviewed in ref. 11) .
  • IL-12 induces cytokine secretion in T lymphocytes and natural killer (NK) cells, increases the cytotoxicity of CTL and NK cells, and promotes the generation of CTL (ref. 12) . Since IL-12 primes CD4 + T cells to produce high levels of ⁇ interferon (IFN ⁇ ) , it is considered to be a primary determinant of T H l-associated immune responses (ref. 13) .
  • the hematopoietic growth factor GM-CSF known to stimulate the proliferation and maturation of APCs (ref. 14), has been linked to augmentation of both anti-tumour and anti-viral immune responses.
  • Enhanced anti-tumour activity was observed when the GM-CSF gene was transduced into tumour cell immunogens (ref. 15), and also when injected as fusion protein, consisting of GM-CSF and a tumour-specific immunoglobulin idiotype (ref. 16) .
  • the immune response generated may not be optimal for all encoded antigens however, it would be useful to provide immunogenic compositions comprising nucleic acid molecules encoding antigens and co-stimulatory molecules and methods for their use for producing an enhanced immune response in a host to the antigen for the production of immunogenic compositions, including vaccines, and diagnostic reagents.
  • the present invention is directed towards the provision of nucleic acid molecules encoding antigens and co-stimulatory molecules and methods of their administration for producing immune responses.
  • a nucleotide vector comprising a first portion having a sequence encoding at least one antigen, a second portion having a sequence encoding at least one co-stimulatory molecule and a promoter operatively coupled to each of said first and second portions for expression of said at least one antigen and said at least one co-stimulatory molecule, including B7-1 and B7-2.
  • the antigen may be an antigen from a pathogen and may be selected from the group consisting of viruses, bacteria and parasites.
  • the pathogen may be an influenza virus and the antigen may be selected from the group consisting of structural and non-structural influenza virus antigens including haemagglutinin, neuraminidase, nucleoprotein, NS1 and NS2.
  • the antigen may be a tumour-associated antigen including carcinoembryonic antigen (CEA) , mutated tumor suppressor ⁇ enes, such as p53, mutated oncogenes, such as ras, or idiotypic markers for tumors, such as B-cell lymphona.
  • CEA carcinoembryonic antigen
  • the vector may further comprise a third portion having a sequence encoding at least one cytokine, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12), interleukin-4 (IL-4) and fragments thereof retaining cytokine activity.
  • cytokine such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12), interleukin-4 (IL-4) and fragments thereof retaining cytokine activity.
  • the vectors of the present invention may be formulated as vaccines for in vivo administration to a host to protect said host against disease caused by said pathogen.
  • a composition comprising the vector described herein and a second nucleotide vector comprising a first portion having a sequence encoding at least one cytokine and a promoter operatively coupled to said first portion of said second vector for expression of said cytokine.
  • the cytokine may be granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-12, interleukin-4 and fragments thereof retaining cytokine activity.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the invention also extends in a particular aspect to a method of generating an immune response in a host comprising administering to the host an effective amount of a first nucleic acid molecule encoding at least one antigen, a second portion nucleic acid molecule encoding at least one co-stimulatory molecule and a promoter operatively coupled to each of said first and second nucleic acid molecules for expression of said at least one antigen and said at least one co-stimulatory molecule.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the first nucleic acid molecule, the second nucleic acid molecule and the promoter preferably are combined in a single nucleotide vector.
  • the antigen may be an antigen from a pathogen (including bacteria, viruses and parasites) or a tumour-associated antigen.
  • the immune response may be a cytotoxic T-cell immune response, and may confer protection to the host against disease caused by the pathogen or a therapeutic immune response against tumour cells.
  • Vectors of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intramuscularly, intradermally or to a mucosal surface of a host, such as by intranasal administration.
  • the method of generating an immune response may further comprise the step of administering to the host a third nucleic acid molecule encoding at least one cytokine and a promoter operatively coupled to the third nucleic acid molecule gene for expression of the at least one cytokine.
  • the cytokine may be macrophage colony stimulating factor (GM-CSF) , interleukin-12, interleukin-4 and fragments thereof retaining cytokine activity.
  • GM-CSF macrophage colony stimulating factor
  • the cytokine gene may be contained within the same vector as the genes encoding the antigens and the co-stimulatory molecules.
  • the at least one third nucleic acid molecule encoding the at least one cytokine may be contained within a second nucleotide vector.
  • the invention extends to a method of using a first gene encoding an antigen and a second gene encoding a co-stimulatory molecule to produce an immune response in a host, comprising the steps of constructing a nucleotide vector comprising the first and second genes, operatively coupling to each of said first and second genes a control sequence to direct expression of the first and second genes in the host, and introducing the vector into the host.
  • the present invention also includes a method for producing an immunogenic composition for evoking a specific immune response to an antigen in a host to which the immunogenic composition is administered, comprising the steps of constructing a nucleotide vector comprising a first gene encoding the antigen and a second gene encoding a co-stimulatory molecule, operatively coupling to each of said first and second genes a control sequence to direct expression of said first and second genes in said host, and formulating said vector as the immunogenic composition for in vivo administration to the host.
  • the antigen may be an antigen of a pathogen and the immunogenic composition may then be formulated as a vaccine for administration to the host to protect the host against disease caused by the pathogen.
  • the invention further includes the use of the nucleotide vector or composition as a medicine.
  • the invention additionally includes the use of the nucleotide vector or composition in the manufacture of a medicament for administration to a host for evoking an immune response to the at least one antigen.
  • Advantages of the present invention include: ease of administration; simplicity of construction; and an increased immune response to the antigen is provided.
  • Figure 1 illustrates the cytotoxic T cell responses in mice immunized with different nucleoprotein gene encoding vectors
  • Figure 2 shows the construction of plasmids for immunization
  • Figure 3 comprising panels a and b, shows the effect upon nucleoprotein-specific CTL responses following injection of co-linear B7-1 and B7-2 plasmids and co-injecting plasmids encoding GM-CSF and IL-12.
  • the present invention relates generally to nucleic acid immunization to produce an immune response (including a protective immune response) by administrating nucleic acid molecules encoding antigens and co-stimulatory molecules, such a B7-1 and B7-2, and, optionally, cytokines, such as GM-CSF, IL-12 and IL-4.
  • NPV gene was cloned into NotI site of pRc/CMV vector (InVitrogen) , and its orientation was determined by restriction digest analysis and sequencing.
  • the NPo gene was amplified from EL4 cells, which had been infected with the influenzae strain X31, by RT-PCR, and was cloned into the pCR3 vector using TA Cloning Kit (InVitrogen) .
  • NP-5' CGCGGCCGCCCGCCATGGCGTCTCAAGGCACC (SEQ ID No: 15); NP-3' : CGTCTAGATTATTAATTGTCGTACTCCTCTGC (SEQ ID No: 16) .
  • the two influenza nucleoprotein (NP) expression vectors (pCMV/NPv and pCMV/NPo) varied greatly in their ability to induce CTL responses (see Figure 1) .
  • the pCMV/NPv vector which encodes a native A/PR/8/34 NP, was better at inducing a CTL response than pCMV/NPo, which expresses a variant NP containing three mutations near the carboxy-terminus of the molecule (ref. 17) .
  • the pCMV/NPv plasmid induced an influenza NP 147-155 peptide-specific CTL response indistinguishable from that obtained from influenza-infected mice, the pCMV/NPo plasmid was unable to induce a CTL response above the pCMV vector control (Fig. 1) .
  • pCMV/NPv and pCMV/NPo also differ in their 5'- and 3'- untranslated regions (UTR) , in that the former retained 5'- and 3'- sequences from the influenza virus UTR, whereas the latter did not.
  • UTR 5'- and 3'- untranslated regions
  • the entire NP coding region and up to 100 nucleotides in the 5'- and 3'- flanking regions were sequenced for both pCMV/NPv and pCMV/NPo, and the following differences found.
  • the pCMV/NPv NP DNA sequence contained two silent mutations, resulting in an amino acid sequence identical to that of A/PR/8/34 (Genbank accession #V01084), whereas pCMV/NPo had three nucleotide mutations leading to amino acid changes F304L, N370S, and G441R.
  • the pCMV/NPv expression vector contained an influenza virus UTR upstream from the translation initiation methionine codon
  • the differences in CTL responses are unrelated to the K d -restricted epitope NP aa 147 to 155, which is identical in each case.
  • the pCMV/NPo vector provided a sub-optimal immunization plasmid which could be used to demonstrate the enhancement of the immunogenicity of an immunogen of the present invention.
  • co-stimulatory molecules are expressed on the same cells displaying the non-self, class I MHC-restricted epitope engaged by the precursor CTL.
  • Co-linear plasmid immunization vectors were constructed for the simultaneous expression of B7-1 or B7-2 in the context of NPo (see Fig. 2) .
  • B7-1 or B7-2 Fluorescence-activated flow cytometry on transiently transfected COS cells was used in each case to establish that B7-1 or B7-2 was expressed at comparable levels on the surface of the relevant transfectants.
  • B7-1 and B7-2 gene products were confirmed to be functional using an in vi tro co-stimulation assay.
  • Vectors were also constructed for (a) the expression of GM-CSF, (b) the co-expression of the p35 and the p40 subunits of ⁇ L-12 in tandem, and (c) expression of the GM-CSF and IL-12 subunits on the same vector (Fig.2) . Enzyme-linked immunosorbent assays and a bioassay were used to confirm the expression of the cytokines.
  • constructs were assayed through transient transfections into COS-7 cells. 48 hours following transfection, cells transfected with B7-1 or B7-2 containing constructs were stained for surface expression of the respective molecules, using the anti-B7-l MAb 1G10, or the anti-B7-2 MAb GLl (Pharmingen, San Diego, CA) . The samples were analyzed on a FACScan flow cytometer (Becton Dickinson) . The supernatants from GM-CSF and/or IL-12 transfected cells were tested for the presence of cytokine five days post-transfection. GM-CSF was detected using a mouse GM-CSF ELISA kit (Endogen, Cambridge, MA) .
  • a bioassay was used which is based on the ability of IL-12 to induce IFN ⁇ production in splenocytes.
  • Supernatants from IL-12 transfected COS-7 cells were added to lxlO 7 mouse splenocytes in serial dilutions (with the addition of 50U of human rIL2/mL) . After a 48 hour incubation, the supernatants were tested for the presence of IFN ⁇ , using a mouse IFN ⁇ ELISA kit (Endogen) .
  • mice received booster injections at three and six weeks. CTL activity was measured two weeks after each booster injection.
  • the data were normalized to the CTL response observed with spleen cells from influenza virus infected mice, the positive control included in each assay.
  • Fig. 3 Even after a second boost with lOO ⁇ g of the cytokine encoding plasmids, the level of lysis does not exceed 20% of the influenza virus control.
  • the NPo plasmid was co-injected with either the GM-CSF or the IL-12 plasmid, no enhancement of the NP specific CTL response was observed after the first boost (Fig. 3A) .
  • anti-NP CTL responses were dramatically increased with both the GM-CSF and IL-12 constructs (Fig. 3B) .
  • NP epitopes generated in the context of B7-2 co-stimulation are better able to take advantage of the GM-CSF and/or IL-12 in their immediate environment to produce a stronger CTL response.
  • Antigens to which a cytotoxic T cell response is desired may be of poor immunogenicity.
  • tumour associated antigens which may differ from self antigens by as little as single point mutations (ref. 19) can be expected to be poorly immunogenic.
  • CTL responses to defined tumour associated antigens are known to be of therapeutic benefit (ref. 20)
  • this situation represents a clear example where the augmentation of in vivo CTL responses to weak, but defined CTL determinants may be of significant benefit.
  • a further practical advantage is the reduction in the amount of plasmid DNA required to :-e injected for even the strongest antigens to invoke a beneficial immune response. This may be particularly important with respect to safety concerns about the risk of plasmid DNA integration into genomic DNA, and the induction of anti-DNA antibodies (ref. 21) .
  • co-stimulatory molecules and cytokines for a particular plasmid immunogen may vary depending on the type of immune response desired.
  • the nature of the co-stimulatory molecule i.e. B7-1 or B7-2
  • T H 1 versus T H 2 balance of the induced response i.e. B7-1 or B7-2
  • cytokines during the initiation of a T cell response can have a dramatic impact on the immunological direction of a particular response.
  • IL-12 is known to promote the development of CTL responses and the T H 1 pathway, whereas IL-4 greatly favours T 2-governed responses (ref. 22) .
  • Immunogenic compositions suitable to be used as vaccines, may be prepared from the vectors as disclosed herein.
  • the vaccine elicits an immune response in a subject.
  • Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically-acceptable liquid solutions or emulsions for polynucleotide administration.
  • the nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 93/24640, (ref. 23)) or the nucleic acid may be associated with an adjuvant, as described in more detail below.
  • Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to 100% of the polynucleotide.
  • polyanions such as DNA and RNA
  • the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment.
  • Published PCT application WO 94/27435 describes compositions for genetic immunization comprising cationic lipids and polynucleotides.
  • Agents which assist in the cellular uptake of nucleic acid such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.
  • Polynucleotide immunogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles.
  • U.S. Patent 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vendels Supra Mole vides (BVSM) .
  • the particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.
  • U.S. Patent 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide) .
  • poly(glycolide) poly(DL-lactide-co-glycolide) , copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone) , poly(esteramides) , polyorthoesters and poly (8-hydroxybutypric acid), and polyanhydrides.
  • WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens.
  • the microspheres being of starch, gelatin, dextran, collagen or albumin.
  • This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa.
  • the delivery vehicle may additionally contain an absorption enhancer.
  • the vectors described herein may be mixed with pharmaceutically acceptable excipients which are compatible therein. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • immunogenic compositions and vaccines comprising the vectors described herein, may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pre-treatment of the injection site with a local anesthetic.
  • the immunogenic compositions formed according to the present invention may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.
  • binders and carriers may include, for example, polyalkalene glycols or triglycerides.
  • formulations may include normally employed incipients, such as for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions can take the form of solutions, suspensions, capsules or sustained release formulations and may contain about 1 to 95% of the vectors described herein .
  • the immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual to synthesize the encoded antigen and mount an immune response. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 ng to about 1 mg of the vectors. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of the host.
  • a vaccine which protects against only one pathogen is a monovalent vaccine.
  • Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention.
  • Such combined vaccines contain, for example, genes encoding antigens from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
  • Immunogenicity can be significantly improved if the vectors are co-administered with adjuvants, commonly used as 0.05 to 0.1 percent solution phosphate-buffered saline.
  • adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves.
  • Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system.
  • Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
  • the vector comprising a first nucleotide sequence encoding at least one antigen and a second nucleic acid molecule encoding at least one co-stimulatory molecule may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immune system.
  • the polynucleotide vectors of the invention may be delivered to the host by a variety of procedures including injection, scarafication, mucosal (for example intranasal) administration, Tang et al. (ref. 24) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice, while Furth et al. (ref. 25) showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals.
  • BGH bovine growth hormone
  • This Example describes CTL responses induced by immunization with different influenza NP-containing vectors.
  • mice were immunized with lOO ⁇ g of pCMV/NPv, pCMV/NPo or vector control pCMV, followed by boosting with lOO ⁇ g of the same, plasmid 3 and 6 weeks later. Spleen cells were obtained and the percent specific lysis was determined in a 4-hour 51 Cr-release. Spleens were harvested 2 weeks after the second boost. Mice more than 4 weeks post-recovery from infection with influenza strain X-31 were used as positive controls (solid square) .
  • Spleen cells from 2 mice in each group were pooled and restimulated for 7 days in vi tro with NP peptide (147-155) -pulsed autologous spleen cells and assayed against NP (147-155) -pulsed P815 cells.
  • This Example describes the construction of immunization plasmids.
  • the B7-1 and GM-CSF genes were amplified from mRNA isolated from LPS-stimulated M12.4.1 cells, and the B7-2 gene was amplified from LPS-stimulated 38C13 cells.
  • the two IL12-encoding genes p35 and p40 were amplified from LPS-stimulated WEHI-3 cells (ATCC TIB 68) .
  • the NP gene was amplified from EL4 cells (ATCC TIB 39) , which had been infected with the influenza strain X-31.
  • the mRNAs were isolated using the Quick prep micro RNA purification kit (Pharmacia, Piscataway, NJ) , and the First-strand cDNA synthesis kit (Pharmacia) was used to synthesize the cDNA.
  • the sequences of the PCR amplifiers were as follows: B7-1-5' : TAT AGC GGC CGC TCC AAA GCA TCT GAA GCT ATG GCT (SEQ ID No: 3); B7-1-3' : TAT AGG GCC CAC AGA GAA GAA CTA AAG GAA GAC (SEQ ID No: 4); B7-2-5': TAT AGC GGC CGC GTT CCA GAA CTT ACG GAA (SEQ ID No: 5); B7-2-3* TAT AGG GCC CAC TGA ACA GTT CTG TGA CAT (SEQ ID No: 6) ; GM-CSF-5 ' : TAT AGC GGC CGC CTC AGA GAG AAA GGC TAA GGT (SEQ ID No: 7); GM-CSF-3' : TAT AGG GCC CTA TCT CTC GTT TGT CTT CCG (SEQ ID No: 8); P35-5': TAT GCG GCC GCG GTC CAG CAT GTG
  • PCR products were initially ligated into the expression vector pcDNA3 (invitrogen, San Diego, CA) , sequenced and tested for expression by various in vitro assays (Fig. 2a) .
  • Fig. 2a in vitro assays
  • they were assayed through transient transfections into COS-7 cells.
  • 48 hours following transfection cells transfected with B7-1 or B7-2 containing constructs were stained for surface expression of these respective molecules, using the anti-B7-l MAb 1G10, or the anti-B7-2 MAb GL1 (Pharmingen, San Diego, CA) .
  • the samples were analyzed on a FACScan flow cytometer (Becton Dickinson) .
  • GM-CSF and/or IL-12 transfected cells were tested for the presence of cytokine five days post transfection.
  • GM-CSF was detected using a mouse GM-CSF ELISA kit (Endogen, Cambridge, MA) .
  • a bioassay was designed which is based on the ability of IL-12 to induce IFN ⁇ production in splenocytes. Briefly, supernatants from IL-12 transfected COS-7 cells were added to 1X10 7 mouse splenocytes in serial dilutions (with the addition of 50U of human rIL2/ml) . After a 48 hour incubation, the supernatants were tested for the presence of IFN ⁇ , using a mouse IFN ⁇ ELISA kit (Endogen) .
  • the pGCVII plasmid (5 Prime-3 Prime Inc., Boulder, CO) was used as the backbone for the immunization vectors.
  • CMV cytomegalovirus
  • BGH bovine growth hormone
  • B7-1 and B7-2 cassettes were removed from Bglll/Nael digestion and ligated into ' the BamHI/Nael sites of plasmid pGCVII (to provide plasmids pGCV.7-1 and pGCV.7-2) .
  • the NP expression cassette was removed with an NruI/PvuII digest, and inserted into the Pmel sites of the plasmid pGCVII, pGCV.7-1 ⁇ and pGCV.7-2 vectors (to provide plasmids pGCV.NPo, pGCV.NPo/7-1 and pGCV.NPo/7-2 respectively) .
  • the GM-CSF cassette was liberated by Seal and PvuII digestion, and inserted into MscI site of plasmid pGCVII (to produce plasmids pGCV.GM-CSF) .
  • the p35 and p40 genes were cloned into the pcDNA3 vector separately.
  • the p35 expression cassette was then removed by an NruI/PvuII digest and inserted into the Nrul site of the p40 plasmid.
  • the p35 and p40 expression units were finally excised on a single fragment by Seal and Drain digestion and inserted into either the MscI site of plasmid pGCVII (to provide plasmid pGCV.IL12) or the Pmel site of the pGCV.GM-CSF plasmid (to provide plasmid pGCV.GM/IL12) .
  • Example 3 This Example describes the NP-specific CTC response in mice immunized with nucleic acid molecules encoding
  • NP NP, B7-1, B7-2, GM-CSF and IL-12 using the vectors described in Example 2.
  • mice were injected with either lOO ⁇ g of the NPo vector pGCV.NPo alone, or lOO ⁇ g of the plasmid in which
  • NPo was co-linear with B7-1 (plasmid pGCV.NPo/B7-l) or B7-2 (plasmid pGCV.NPo/B7-2) .
  • Each plasmid was also co- injected with lOO ⁇ g of the plasmids encoding GM-CSF (pGCV.GM-CSF) , IL-12 (pGCV.ILl2), or the GM-CSF/IL-12 co-linear construct (pGCV.GM/IL12) in the hind leg muscle on 0, 3 and 6 weeks.
  • Splenocytes were harvested 2 weeks after the first boost (Fig. 3, panel a) or 2 weeks after the second boost (Fig.
  • the present invention provides certain vectors and methods for generating immune responses by co-administration of nucleotide vectors encoding antigens and co-stimulatory molecules.
  • the immunization may also include administration of at least one cytokine-encoding gene. Modifications are possible within the scope of this invention.

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Abstract

La présente invention, qui concerne des vecteurs nucléotides codant pour des antigènes, concerne également des molécules de co-stimulation telles que les B7-1 (CD80) ou B7-2 (CD86). Ces vecteurs sont administables à un hôte selon diverses voies en vue de provoquer une réponse immunitaire. Les antigènes concernés sont des antigènes d'origine pathogène et des antigènes associés à un phénomène tumoral. En l'occurrence, la réponse immunitaire peut être une réponse cytotoxique de lymphocytes T, et, si l'antigène est d'origine pathogène, il est capable de produire une protection contre une affection provoquée par un agent pathogène. Pour augmenter encore plus la réponse immunitaire, on peut également administrer, non seulement une cytokine codant pour un gène, telle que le facteur stimulant les colonies de granulocytes-macrophages (GM-CSF), mais aussi l'interleukine-12 (IL-12). De tels vecteurs conviennent aux interventions vaccinales et thérapeutiques contre des affections infectieuses ou néoplasiques.
PCT/CA1997/000162 1996-03-08 1997-03-07 Procede et compositions nucleiques immunogenes codant pour des antigenes, et molecules co-stimulant l'immunisation WO1997032987A1 (fr)

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WO1998044788A3 (fr) * 1997-04-09 1999-03-11 Chang Lung Ji Modele animal aux fins d'une appreciation de vaccins
WO1999036089A1 (fr) * 1998-01-16 1999-07-22 The Johns Hopkins University Immunisation genetique par l'administration conjointe d'acides nucleiques et de cytokines dans un excipient unique
WO1999041368A3 (fr) * 1998-02-11 1999-12-16 Maxygen Inc Optimisation des proprietes immunomodulatrices des vaccins genetiques
EP1131432A2 (fr) * 1998-11-18 2001-09-12 Pacific Northwest Research Institute Vaccins a base d'antigenes recepteurs de surface
WO2001032204A3 (fr) * 1999-11-03 2002-07-25 Powderject Vaccines Inc Composition vaccinale a base d'acides nucleiques comportant un promoteur de gene cd80/cd86 mammalien suscitant une expression antigenique
WO2002057452A3 (fr) * 2000-12-15 2003-08-21 Curagen Corp Proteines, polynucleotides codant pour elles et leurs procedes d'utilisation
US6730512B2 (en) 1997-04-09 2004-05-04 Amdl, Inc. Combination immunogene therapy
US6881723B1 (en) 1998-11-05 2005-04-19 Powderject Vaccines, Inc. Nucleic acid constructs
EP1518927A3 (fr) * 1998-02-11 2005-06-22 Maxygen, Inc. Optimisation des propriétés immunomodulatrices de vaccins génétiques
US7022320B1 (en) 1999-02-09 2006-04-04 Powderject Vaccines, Inc. Mycobacterium tuberculosis immunization
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US7223739B1 (en) 1995-06-07 2007-05-29 Powderject Vaccines, Inc. Adjuvanted genetic vaccines
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
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Cited By (16)

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US7699801B2 (en) 1995-06-07 2010-04-20 Powderject Vaccines, Inc. Advanced genetic vaccines
US7223739B1 (en) 1995-06-07 2007-05-29 Powderject Vaccines, Inc. Adjuvanted genetic vaccines
WO1998044788A3 (fr) * 1997-04-09 1999-03-11 Chang Lung Ji Modele animal aux fins d'une appreciation de vaccins
US6730512B2 (en) 1997-04-09 2004-05-04 Amdl, Inc. Combination immunogene therapy
AU743226B2 (en) * 1998-01-16 2002-01-24 Johns Hopkins University, The Genetic immunization with co-delivery of nucleic acid and cytokines in a single vehicle
WO1999036089A1 (fr) * 1998-01-16 1999-07-22 The Johns Hopkins University Immunisation genetique par l'administration conjointe d'acides nucleiques et de cytokines dans un excipient unique
EP1518927A3 (fr) * 1998-02-11 2005-06-22 Maxygen, Inc. Optimisation des propriétés immunomodulatrices de vaccins génétiques
WO1999041368A3 (fr) * 1998-02-11 1999-12-16 Maxygen Inc Optimisation des proprietes immunomodulatrices des vaccins genetiques
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
US6881723B1 (en) 1998-11-05 2005-04-19 Powderject Vaccines, Inc. Nucleic acid constructs
EP1131432A2 (fr) * 1998-11-18 2001-09-12 Pacific Northwest Research Institute Vaccins a base d'antigenes recepteurs de surface
US7022320B1 (en) 1999-02-09 2006-04-04 Powderject Vaccines, Inc. Mycobacterium tuberculosis immunization
WO2001032204A3 (fr) * 1999-11-03 2002-07-25 Powderject Vaccines Inc Composition vaccinale a base d'acides nucleiques comportant un promoteur de gene cd80/cd86 mammalien suscitant une expression antigenique
US7196066B1 (en) 1999-11-03 2007-03-27 Powderject Vaccines, Inc. DNA-vaccines based on constructs derived from the genomes of human and animal pathogens
WO2002057452A3 (fr) * 2000-12-15 2003-08-21 Curagen Corp Proteines, polynucleotides codant pour elles et leurs procedes d'utilisation
WO2009044165A3 (fr) * 2007-10-05 2009-06-11 Isis Innovations Ltd Adjuvant moléculaire

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