EP1599581A2 - Production d'un vaccin vivant agissant contre des virus de la grippe - Google Patents

Production d'un vaccin vivant agissant contre des virus de la grippe

Info

Publication number
EP1599581A2
EP1599581A2 EP03773534A EP03773534A EP1599581A2 EP 1599581 A2 EP1599581 A2 EP 1599581A2 EP 03773534 A EP03773534 A EP 03773534A EP 03773534 A EP03773534 A EP 03773534A EP 1599581 A2 EP1599581 A2 EP 1599581A2
Authority
EP
European Patent Office
Prior art keywords
virus
influenza virus
elastase
protease
attenuated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03773534A
Other languages
German (de)
English (en)
Inventor
Hans-Dieter Klenk
Jürgen STECH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Transmit Gesellschaft fuer Technologietransfer mbH
Philipps Universitaet Marburg
Original Assignee
Transmit Gesellschaft fuer Technologietransfer mbH
Philipps Universitaet Marburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Transmit Gesellschaft fuer Technologietransfer mbH, Philipps Universitaet Marburg filed Critical Transmit Gesellschaft fuer Technologietransfer mbH
Publication of EP1599581A2 publication Critical patent/EP1599581A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • 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
    • C12N2760/16122—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
    • 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
    • C12N2760/16161—Methods of inactivation or attenuation

Definitions

  • the object of the present invention is to eliminate the disadvantages described in the prior art and to provide a safe method for producing new immunogenic but not pathogenic mutants of influenza viruses, the genome of which largely corresponds to that of the epidemic strain, which is living as attenuated recombinant -Vaccines can also be used in children, the elderly and immunodeficient people against the influenza virus.
  • FIG. 2 shows the results of the plaque test of the elastase-dependent recombinant virus mutant in the presence of elastase (A) and in the presence of trypsin (B).
  • the plasmid pHW184-HA contains the HA and is the target of the mutation.
  • the nucleotides AG are exchanged for GT at positions 1027 and 1028 in the coding region of the HA gene of the strain A / WSN / 33 (Accession J02176) by means of PCR technology. This leads to the replacement of the amino acid arginine with valine.
  • the recombinant influenza virus with this mutation is hereinafter referred to as WSN-E, the unmodified recombinant wild-type virus as WSNwt.
  • any transfection protocol known in the prior art is suitable.
  • elastase 5 ⁇ g / ml, elastase from pig pancreas, Sigma
  • the conditions for further virus replication after transfection can be individually adapted to the requirements of the virus to be released. Depending on the properties of the starting isolate, this is easily possible for the person skilled in the art.
  • a combination with other attenuating, for example temperature-sensitive mutations leads to the production of a multi-attenuated virus.
  • additional elastase is added to the cell culture at a correspondingly reduced temperature.
  • a cytopathic effect in the form of morphological cell changes is typically visible on the tissue culture cells, the cells lose their shape, they become smaller and detach, and cell contacts are lost.
  • the mutations were confirmed with sequence analysis and the protease dependency was demonstrated in the plaque test, with the corresponding protease, here the elastase, being added instead of the commonly used trypsin.
  • FIGs 5, 6 and 7 summarize the results of an immunization experiment in mice in which the mice are inoculated intranasally. This immunization is well tolerated with no symptoms of illness or weight loss (data not shown).
  • Figure 5 shows that all mice survive intranasal immunization with the elastase-dependent recombinant virus WSN-E.
  • the experiments are carried out in three groups: 10 6 pfu WSN-E inactivated with formalin (marked with a cross), 10 3 pfu WSN-E as live virus (marked with a dot) and 10 6 pfu WSN-E as live virus (marked with a square) ,
  • FIG. 7 shows the result of the plaque test with lung homogenate.
  • WSN-E in a lower dose of 10 3 pfu and non-immunized mice as a positive control, plaque titres in the lungs can be clearly detected, whereas in mice in the group with WSN-E in high dose of 10 6 pfu had been immunized, no plaque titer was detectable in the lungs.
  • the recombinant viruses are produced, for example, using the plasmid-based system of reverse genetics from Hoffmann et al., (PNAS 97, 6108-6113 (2000).
  • PNAS 97, 6108-6113 a system of reverse genetics from Hoffmann et al.
  • all 8 gene segments must be amplified by RT-PCR and in the plasmid vector pHW2000 can be cloned (Hoffmann et al., PNAS, Vol. 97, 6108-6113, 2000).
  • Any starting material eg chicken egg allantoic fluid or cell culture supernatants
  • This methodology is familiar to the person skilled in the art.
  • cultures (5 ml) are inoculated in LB medium overnight and the plasmid DNA is isolated using a suitable method, for example using the Qiaprep8-Miniprep TM kit (from Qiagen, Hilden, Germany). The clones are checked for correct mutation by sequencing. Positive clones with the desired mutation are expanded using the Qiafilter Plasmid Maxi TM kit from Qiagen or a similar method in order to have plasmid DNA in the microgram range per microliter available for the transfections. 2. Virus release

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Pulmonology (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un nouveau procédé de production d'un virus atténué de la grippe, homologue à la souche de type sauvage épidémique, utilisé comme vaccin contre le virus de la grippe. Suite à la modification d'une protéine superficielle de la grippe, le virus perd son pouvoir pathogène, sans perdre pour autant son pouvoir immunogène et s'utilise comme vaccin vivant dans la vaccination d'individus, notamment d'enfants, de personnes âgées et de personnes immunodéficientes.
EP03773534A 2002-10-16 2003-10-13 Production d'un vaccin vivant agissant contre des virus de la grippe Withdrawn EP1599581A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10248301 2002-10-16
DE10248301A DE10248301A1 (de) 2002-10-16 2002-10-16 Herstellung eines Lebend-Impfstoffes gegen Influenza-Viren
PCT/DE2003/003420 WO2004034956A2 (fr) 2002-10-16 2003-10-13 Production d'un vaccin vivant agissant contre des virus de la grippe

Publications (1)

Publication Number Publication Date
EP1599581A2 true EP1599581A2 (fr) 2005-11-30

Family

ID=32049328

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03773534A Withdrawn EP1599581A2 (fr) 2002-10-16 2003-10-13 Production d'un vaccin vivant agissant contre des virus de la grippe

Country Status (4)

Country Link
EP (1) EP1599581A2 (fr)
AU (1) AU2003281953A1 (fr)
DE (1) DE10248301A1 (fr)
WO (1) WO2004034956A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004013335A1 (de) * 2004-03-17 2005-10-13 TransMIT Gesellschaft für Technologietransfer mbH Impfstoff gegen Influenza basierend auf Geflügelpestviren
WO2011151470A2 (fr) * 2010-06-02 2011-12-08 Avir Green Hills Biotechnology Research Development Trade Ag Nouveau procédé de génération d'un virus à arn
KR102748840B1 (ko) 2016-02-03 2025-01-02 씨지 디스커버리, 인코포레이티드 이종 에피토프 및/또는 성숙 절단 부위를 갖는 인플루엔자 헤마글루티닌 조성물
US10063211B2 (en) 2016-02-03 2018-08-28 Qualcomm Incorporated Compact bypass and decoupling structure for millimeter-wave circuits

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL205955B1 (pl) * 2000-04-28 2010-06-30 St Jude Childrens Res Hospital Układ oparty na minimalnej liczbie plazmidów do wytwarzania infekcyjnych wirusów RNA o ujemnej nici, komórka gospodarza zawierająca taki układ, sposób wytwarzania wiriona wirusa RNA o ujemnej nici, sposób wytwarzania atenuowanego wirusa RNA o ujemnej nici, sposób wytwarzania zakaźnego wirusa o ujemnej nici do stosowania w szczepionkach i zastosowanie układu opartego na minimalnej liczbie plazmidów

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004034956A2 *

Also Published As

Publication number Publication date
DE10248301A1 (de) 2004-04-29
AU2003281953A1 (en) 2004-05-04
WO2004034956A2 (fr) 2004-04-29
AU2003281953A8 (en) 2004-05-04
WO2004034956A3 (fr) 2004-07-15

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