WO2003020322A1 - Vecteur d'induction d'une reponse immune - Google Patents

Vecteur d'induction d'une reponse immune Download PDF

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Publication number
WO2003020322A1
WO2003020322A1 PCT/EP2002/009909 EP0209909W WO03020322A1 WO 2003020322 A1 WO2003020322 A1 WO 2003020322A1 EP 0209909 W EP0209909 W EP 0209909W WO 03020322 A1 WO03020322 A1 WO 03020322A1
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Prior art keywords
virus
vector according
antigen
immune response
viruses
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PCT/EP2002/009909
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German (de)
English (en)
Inventor
Andreas Hüser
Christian Hofmann
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DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
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Publication of WO2003020322A1 publication Critical patent/WO2003020322A1/fr

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    • 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
    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to recombinant vectors comprising a non-mammalian DNA virus which express one or more antigens and their use for inducing an immune response in mammals, in particular in humans.
  • the induction of an immune response is the basis for the production of antibodies and vaccines.
  • the protein (antigen) against which an immune response is to be achieved is produced recombinantly and then applied in combination with an adjuvant. This method is very complex since it is often difficult to produce the antigen sufficiently in its natural protein structure.
  • the use of attenuated bacteria or viruses as carriers for the expression of heterologous antigens is known.
  • a disadvantage of this live vaccine is that the pathogen used as the carrier can cause undesirable effects.
  • non-mammalian DNA viruses e.g. WO-A-95/23866 or WO96 / 09074.
  • coat protein-modified non-mammalian DNA virus vectors are known, for example from WO99 / 091 93 and US Patents 6, 1 83.993 and 6, 1 90.887. Reference is expressly made to the disclosure of these documents with regard to suitable viral vectors or the modification of the coat protein.
  • the present application describes the invention that an unexpectedly strong immune response against antigens is generated when these respond to the Surface of a non-mammalian DNA virus, such as the baculovirus, brought. Furthermore, it was discovered that the expression of a heterologous DNA sequence also results in a strong immune response against the product of the synthesized protein.
  • Possible applications of the invention are, for example, the production of antibodies, the vaccination against pathogens or tumors and the isolation of immune response-inducing proteins and others.
  • a first aspect of the invention relates to a recombinant vector comprising a non-mammalian DNA virus which has at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells, e.g. Promoter, and optionally enhancer, contains.
  • a heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells, e.g. Promoter, and optionally enhancer, contains.
  • the heterologous DNA sequence can code for a product against which an immune response is to be induced or / and for a product which enhances an immune response (directed against another product).
  • the DNA virus may optionally be a modified envelope, e.g. as described in one of the aforementioned documents.
  • the envelope is particularly preferably modified by expression of antigens in order to further strengthen the immune response.
  • a suitable promoter active in mammalian cells e.g. Rous-Sarcoma Virus LTR (RSV) promoter or cytomegalovirus (CMV) promoter.
  • Another aspect relates to a recombinant vector comprising a non-mammalian DNA virus, which is at least one heterologous, for an antigen encoding DNA sequence, fused with a DNA sequence coding for a coat protein of the virus.
  • Baculovirus gp64 protein or a corresponding coat protein from another virus can be used as suitable coat proteins which can be fused with the antigen.
  • the heterologous DNA sequence can be fused to the viral sequence coding for a coat protein, C-terminally, N-terminally and / or inside the viral sequence.
  • Yet another aspect of the invention relates to a recombinant vector comprising a non-mammalian DNA virus, the at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells and at least one heterologous DNA coding for an antigen Sequence, fused to a DNA sequence coding for a coat protein of the virus.
  • Preferred embodiments of the vector according to the invention are the subject of dependent claims 4 to 10.
  • the invention also relates to a pharmaceutical composition which contains as an active ingredient a viral vector according to the invention together with pharmaceutically customary carriers, auxiliaries and diluents.
  • the composition may further contain a mammalian immune response-enhancing adjuvant, such as aluminum hydroxide or cytosine / guanine-rich sequences.
  • composition is suitable for use as a vaccine, for inducing an immune response in mammals, for producing antigens and for isolating proteins which induce an immune response.
  • the composition can be administered by any means, for example by injection, for example subcutaneous, intramuscular or intraperitoneal injection, by oral administration, by inhalation or nasal administration or by any other suitable administration method.
  • the administration can take place in one or preferably in several doses.
  • the dose amount is preferably 10 7 to 10 9 , in particular 10 8 vector units / kg.
  • the viral vectors according to the invention are produced by customary methods, with plasmids carrying the corresponding heterologous DNA sequences first being produced as recombination vectors and transfected together with the DNA of the non-mammalian virus in permessive cells and the recombinant vector being obtained by homologous recombination.
  • the invention relates to a vector which induces a specific immune response against products of inserted sequences in mammals and / or mammalian cells. Areas of application are e.g. medicine, biotechnology and genetic engineering.
  • the vector preferably consists of an insect virus, preferably a representative of the baculoviruses or a nuclear polyhedrosis virus, which contains at least one component selected from (i) a modified envelope, (ii) a heterologous DNA sequence and (iii) one for the Gene expression suitable promoter.
  • Table 1 shows the detection of the induction of a strong immune response by expression and / or surface presentation.
  • Baculoviruses generated using psCSvac, peCSvac and psCSeCS-vac were injected into BalbC mice and the immune response against the antigen was determined using the antibody in an ELISA test (after 56 days).
  • Table 2 shows the ratio of immunoglobulins G 1 and G2a (IgG 1 / IgG2a) in mouse serum. This ratio was obtained from the data in Figures 9 and 10. Cytokines regulate the production of classes and subclasses of antibodies. Therefore, a low ratio of IgG 1 / IgG2a indicates a cellular immune response. The low ratio of IgG1 / IgG2a after immunization with the Baculovirus AcNPVsCS / eCS gives a clear indication of a cellular immune response. In contrast, immunization with recombinant CS protein and Alhydrogel ® shows a high IgG1 / IgG2a ratio, which does not indicate a cellular immune response.
  • Figure 1 shows the recombination vector psCSvac.
  • the antigen here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum
  • CS circumsporozoite protein
  • Figure 2 shows the recombination vector peCSvac.
  • the antigen here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum
  • CS circumsporozoite protein
  • Figure 3 shows the recombination vector psCS / eCSvac.
  • the antigen here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum
  • Figure 4 shows a schematic of the new vectors.
  • psCS results in a vector that carries the antigen (s) on the surface.
  • peCSvac results in a vector that expresses the antigen (s) in the cell.
  • psCS / eCSvac results in a vector which carries the antigen (s) on the surface and at the same time expresses the antigen (s) in the cell.
  • Figure 5 shows the evidence of induction of a strong immune response.
  • a baculovirus which was generated by means of the vector psCSvac, was injected into BalbC mice and mediated a strong and long-lasting immune response against the CS antigen, which could be increased by repeated administration (boost).
  • Figure 6 shows an overview of the immunization regime and the animal test conditions of Example 2.
  • Figure 7 shows the malaria CS-specific immunoglobulin M (IgM) immune response.
  • Figure 8 shows the malaria CS-specific total immunoglobulin G (IgG H + L, heavy and light chain) immune response.
  • Figure 9 shows the malaria CS-specific immunoglobulin G1 (IgGD immune response.
  • Figure 10 shows the malaria CS-specific immunoglobulin G2a (lgG2a) immune response.
  • Figure 1 1 shows the malaria CS-specific cellular immune response from Baculovirus AcNPVsCS / eCS.
  • Example 1 Preparation of a non-mammalian DNA virus that carries antigens on the surface.
  • a sequence for an N-terminally modified non-mammalian DNA virus coat protein (baculovirus, gp64) was cloned into a recombination vector in a known manner under the control of a baculoviral promoter.
  • the modification was designed by inserting DNA sequences which code for antigens from pathogens, viruses and / or tumors. This sequence is achieved between the signal sequence and the sequence of the baculovirus coat protein gp64 at the DNA level.
  • CS plasmodium . falciparum circumsporozoite protein
  • the recombination vectors were produced using standard methods.
  • the plasmids are shown in Figure 1-3 and can be modified as desired by replacing the CS with other antigens, such as.
  • Recombination vectors are shared with the DNA of one
  • Example 2 Immunization regimes and animal testing conditions.
  • balb / c mice The following agents were injected into balb / c mice, divided into three groups of three animals each under the following conditions:
  • CS malaria Circumsporozoit
  • PBS (mock) injected animals serve as controls.
  • recombinant CS protein was adsorbed on 96-well plates, then the plates were incubated with different dilutions of the mouse sera from the treated animals and subsequently by means of an enzyme-coupled anti-IgM- (Figure 7), anti-IgG- ( Figure 8), anti-lgG 1 ( Figure 9) and anti lgG2a- ( Figure 10) specific antibody quantitatively developed according to standard methods.
  • the spleen was removed after five weeks to determine the specific T cell response in the ELIspot assay (ELI).
  • the ELIspot method detects the specific secretion of interferon-gamma by T cells from the spleen of immunized mice after activation of the T cells with CS protein.
  • T cells from mice immunized with Baculovirus AcNPVsCS / eCS show a clear interferon-gamma secretion in the ELIspot (22 spots at 2 ⁇ g CS, 89 spots at 10 ⁇ g CS antigen (Figure 1 1)).
  • Concanavalin A activates T cells non-specifically and serves as a positive control in this experiment. This shows that the baculovirus is able to induce a strong cellular immune response that is necessary to protect humans from pathogens such as Plasmodium falciparum.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
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Abstract

L'invention concerne des vecteurs recombinants contenant un virus ADN de non mammifère et exprimant au moins un antigène. L'invention concerne également leur utilisation pour induire une réponse immune chez les mammifères, notamment chez l'homme.
PCT/EP2002/009909 2001-09-04 2002-09-04 Vecteur d'induction d'une reponse immune WO2003020322A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01121194.3 2001-09-04
EP01121194 2001-09-04

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WO2003020322A1 true WO2003020322A1 (fr) 2003-03-13

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020236A3 (fr) * 2007-08-07 2009-04-16 Educational Foundation Jichi M Nouveau vecteur viral
US9023365B2 (en) 2006-02-09 2015-05-05 Educational Foundation Jichi Medical University Recombinant baculovirus vaccine
US9327018B2 (en) 2006-02-09 2016-05-03 Educational Foundation Jichi Medical University Recombinant baculovirus vaccine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2700957A1 (fr) * 1993-01-29 1994-08-05 Seppic Sa Composition de vaccin sous-unitaire recombinant vivant et procédé de préparation.
WO1996009074A1 (fr) * 1994-09-23 1996-03-28 The General Hospital Corporation Utilisation d'un virus a adn non mammalien en vue de l'expression d'un gene exogene dans une cellule mammalienne
WO2000077233A2 (fr) * 1999-06-10 2000-12-21 The General Hospital Corporation Virus d'adn non mammalien resistant au complement et utilisations de ces virus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2700957A1 (fr) * 1993-01-29 1994-08-05 Seppic Sa Composition de vaccin sous-unitaire recombinant vivant et procédé de préparation.
WO1996009074A1 (fr) * 1994-09-23 1996-03-28 The General Hospital Corporation Utilisation d'un virus a adn non mammalien en vue de l'expression d'un gene exogene dans une cellule mammalienne
WO2000077233A2 (fr) * 1999-06-10 2000-12-21 The General Hospital Corporation Virus d'adn non mammalien resistant au complement et utilisations de ces virus

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AOKI HIROSHI ET AL: "Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells.", VETERINARY MICROBIOLOGY, vol. 68, no. 3-4, 31 August 1999 (1999-08-31), pages 197 - 207, XP002224593, ISSN: 0378-1135 *
BOUBLIK Y ET AL: "EUKARYOTIC VIRUS DISPLAY: ENGINEERING THE MAJOR SURFACE GLYCOPROTEIN OF THE AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS (ACNPV) FOR THE PRESENTATION OF FOREIGN PROTEINS ON THE VIRUS SURFACE", BIO/TECHNOLOGY, NATURE PUBLISHING CO. NEW YORK, US, vol. 13, no. 10, 13 October 1995 (1995-10-13), pages 1079 - 1084, XP001119233, ISSN: 0733-222X *
GRABHERR R ET AL: "EXPRESSION OF FOREIGN PROTEINS ON THE SURFACE OF AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS", BIOTECHNIQUES, EATON PUBLISHING, NATICK, US, vol. 22, no. 4, April 1997 (1997-04-01), pages 730 - 735, XP001119232, ISSN: 0736-6205 *
HUESER ANDREAS ET AL: "Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors.", NATURE BIOTECHNOLOGY, vol. 19, no. 5, May 2001 (2001-05-01), pages 451 - 455, XP002224592, ISSN: 1087-0156 *
NIWA H ET AL: "EFFICIENT SELECTION FOR HIGH-EXPRESSION TRANSFECTANTS WITH A NOVEL EUKARYOTIC VECTOR", GENE, ELSEVIER BIOMEDICAL PRESS. AMSTERDAM, NL, vol. 108, 1991, pages 193 - 200, XP000569330, ISSN: 0378-1119 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9023365B2 (en) 2006-02-09 2015-05-05 Educational Foundation Jichi Medical University Recombinant baculovirus vaccine
US9327018B2 (en) 2006-02-09 2016-05-03 Educational Foundation Jichi Medical University Recombinant baculovirus vaccine
US9333249B2 (en) 2006-02-09 2016-05-10 Educational Foundation Jichi Medical University Recombinant baculovirus vaccine
WO2009020236A3 (fr) * 2007-08-07 2009-04-16 Educational Foundation Jichi M Nouveau vecteur viral
JP2010535466A (ja) * 2007-08-07 2010-11-25 学校法人自治医科大学 新規ウイルスベクター
RU2491093C2 (ru) * 2007-08-07 2013-08-27 Эдьюкейшнл Фаундейшн Дзити Медикал Юниверсити Бакуловирусные векторы с двойным промотором, включающим в себя промотор позвоночного и промотор бакуловируса, контролирующим иммуногенный слитый ген
AU2008284664B2 (en) * 2007-08-07 2014-05-15 Educational Foundation Jichi Medical University Baculoviral vectors with a dual vertebrate and baculovirus promoter controlling an immunogenic fusion gene
CN101772576B (zh) * 2007-08-07 2016-05-04 学校法人自治医科大学 带有控制免疫原性融合基因的脊椎动物和杆状病毒双重启动子的杆状病毒载体

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