WO2003083095A1 - Mutants viraux manipules dans les sites de clivage par la furine de glycoproteines - Google Patents

Mutants viraux manipules dans les sites de clivage par la furine de glycoproteines Download PDF

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WO2003083095A1
WO2003083095A1 PCT/EP2003/002520 EP0302520W WO03083095A1 WO 2003083095 A1 WO2003083095 A1 WO 2003083095A1 EP 0302520 W EP0302520 W EP 0302520W WO 03083095 A1 WO03083095 A1 WO 03083095A1
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sequence
virus
heterologous
cleavage sites
furin cleavage
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PCT/EP2003/002520
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English (en)
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Gunther Michael Keil
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Akzo Nobel N.V.
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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
    • 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
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Viral mutans manipulated in the furin cleavage sites of glycoproteins.
  • the present invention relates to viruses containing glycoproteins with furin binding sites. More in particular the present invention relates to mutated Bovine Respiratory Syncytial virus (BRSV), vaccines based thereon and methods for the preparation of such vaccines.
  • BRSV Bovine Respiratory Syncytial virus
  • Bovine Respiratory Syncytial virus is a member of the family paramyxoviridae, and belongs to the genus of the Pneumoviruses.
  • Paramyxoviridae belonging to this genus are human RSV, bovine RSV, ovine RSV, caprine RSV and pneumonia virus of mice.
  • RSV is a member of the order Mononegavirales, i.e. the virus is a non-segmented negative strand RNA virus.
  • the overall genomic organization of the non-segmented negative stranded RNA of the viruses belonging to the genus Pneumoviruses is comparable.
  • the RNA consists of 10 genes, encoding eleven proteins, and has a length of about 15.2 kilobases.
  • the RSV- proteins include two non-structural proteins (NS1 and NS2), four RNA-associated proteins, the nucleoprotein N, phosphoprotein P, the large, catalytic subunit L of the RNA-polymerase, a transcription elongation factor encoded by the first of two overlapping open reading frames of the M2 gene, and three envelope-associated proteins; the fusion protein F, the attachment protein G and the small hydrophobic protein SH.
  • NS1 and NS2 non-structural proteins
  • RNA-associated proteins the nucleoprotein N, phosphoprotein P, the large, catalytic subunit L of the RNA-polymerase, a transcription elongation factor encoded by the first of two overlapping open reading frames of the M2 gene, and three envelope-associated proteins; the fusion protein F, the attachment protein G and the small hydrophobic protein SH.
  • NS1 and NS2 non-structural proteins
  • RNA-associated proteins the nucleoprotein N, phosphoprotein P, the large, catalytic sub
  • One way to mimic the natural infection, c.q. to efficiently trigger the host's defense mechanism is to develop a live attenuated vaccine.
  • a vaccine mimics the natural triggering of the immune system, whereas due to its attenuated characteristics, it does not induce the severe clinical signs caused by the wild-type virus.
  • the attenuated character is usually obtained by mutating a gene that on the one hand plays a role in virulence, but on the other hand is not essential for viral infection and replication, and moreover plays no role in the induction of immunity.
  • Live attenuated vaccines must, as closely as possible, mimic the native RSV as far as their infection behavior is concerned.
  • One of the main characteristics of native Respiratory Syncytial Virus is that shortly after infection it causes formation of large syncytia. Formation of these syncytia, the result of large scale cell fusion, requires the presence of the F-, the G- and the SH-protein. (Heminway, B.R. et a!., Virology 200: 801-805 (1994). Co-expression of both the F- and SH-gene, or of both the F- and G-gene gives only very low level cell-fusion (Pastey, M.K. and Samal, S.K., J. Gen. Virol. 78: 1885-1889 (1997)).
  • the fusion (F) protein is responsible for fusion of the viral and cellular membranes. Fusion of the virus and the cell membranes is thought to occur at the surface of the cell. It is necessary for penetration of the cell by the virus and to transfer the viral ribonucleoprotein into the cell cytoplasm. The F protein mediates penetration, but also promotes syncitia formation (fusion of infected cell membranes with those of adjacent cells).
  • the F glycoprotein is synthesized as an F 0 precursor, which is cleaved post-translationally by furin-like proteases during transport to the cell surface. Cleavage at two distinct sites (prior Glyno and after Arg 136 ) by the furin-like proteases is required for generation of fusion active molecules (Zimmer et al., J. Biol. Chem. 276;Gonzales-Reyes et al., PNAS 98). The resulting subunits,F1 and F2, remain linked by disulphide bonds.
  • oligopeptides can be integrated between 2 furin cleavage sites of a carrier glycoprotein, such as the furin cleavage sites of an F protein of an RSV. Due to the processing of the precursor protein, resulting in the cleavage at the furin cleavage sites, the heterologous sequence may be excised and consequently be secreted by a virus-infected cell.
  • This concept can be applied likewise to other glycoproteins containing two furin cleavage sites, where the original sequence between the cleavage sites may be replaced (in part) by a heterologous sequence or may simply be deleted.
  • heterologous sequences may be inserted together with the a sequence for a second cleavage site, thus creating a situation where the heterologous sequence is again flanked by two furin cleavage sites in the recombinant protein. Processing of the mutated protein will than also result in the excision of the heterologous sequence which may be secreted by virus infected cells.
  • viruses with glycoproteins that may be manipulated in such a way are for example bovine herpesvirus 1 or e.g. Newcastle disease virus or ILTV.
  • Viruses wherein glycoproteins are modified in one of the above-described ways can be used for, for example vaccine purposes.
  • the mutations between the furin binding sites may result in an attenuated virus, or may be used to introduce a positive marker into a vaccine or for co-expression of other heterologous sequences.
  • the present invention relates to a virus having a glycoprotein with one or more furin cleavage sites, characterised in that nucleic acid sequence encoding the glycoprotein is mutated in such a way that either at least part of the naturally occurring sequence between the codons encoding two furin cleavage sites has been deleted, and/or a heterologous sequence has been inserted, wherein, if the heterologous sequence is inserted next to a furin binding site, the inserted sequence contains the heterologous sequence linked to the sequence of another furin cleavage site.
  • heterologous sequence a sequence is meant that does not naturally occur in this location in the viral genome (nucleic acid sequence) or in the sequence of the glycoprotein encoded thereby (amino acid sequence).
  • the present invention relates to a Respiratory Syncytial Virus, preferably BRSV characterized in that at least part of the IFCSP has been deleted or replaced by a heterologous amino acid sequence or a heterologous amino acid sequence has been inserted.
  • a deletion is made in the IFCSP and most preferably all of the IFCSP has been deleted.
  • the BRSV can have a deletion only, or, in the alternative, the IFCSP has been deleted and replaced by heterologous sequences.
  • the mutated BRSV according to the invention can be used as attenuated live vaccines.
  • Heterologous sequences, when inserted in the IFCSP locus may encode biological important peptides, parts of proteins, or complete heterologous proteins.
  • the BRSV of the present invention may thus be used in vaccines for the protection of a host against BRSV infection.
  • the recombinant or mutated BRSV according to the invention can be produced by mutating the gene sequence encoding the F protein.
  • Mutations can be introduced by means of standard recombinant DNA technology.
  • Standard recombinant DNA techniques such as making cDNA, cloning of cDNA in a plasmid, digestion of the gene with a restriction enzyme, followed by endonuclease treatment, re-ligation and homologous recombination in the host strain, are all known in the art and described i.a. in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0- 87969-309-6).
  • Site-directed mutations can e.g. be made by means of in vitro site directed mutagenesis using the Transformer® kit sold by Clontech.
  • RSV mutants according to the present invention are very suitable as carriers for heterologous RNA sequences.
  • This heterologous RNA can be inserted in the region that was deleted from the F -gene.
  • a heterologous RNA sequence is a sequence that originates from a source, other than the parental (B)RSV strain. It may be derived from the DNA of another organism or may be synthetically made. In an even more preferred form, the heterologous RNA sequence codes for a polypeptide.
  • the heterologous RNA sequence may contain promotor sequences such that expression is under control of these sequences.
  • promotor sequences may be the promotor sequences that are found to be linked to the heterologous gene coding for the polypeptide, in its native form, or it may be other promotor sequences suitable for expression in eukaryotic cells. It is obvious to those skilled in the art that the choice of a promotor extends to any eukaryotic, prokaryotic, viral or synthetically prepared promotor capable of directing gene transcription in cells infected by the BRSV mutant. Such promotors may be HRSV or BRSV promotors, but also promotors obtained from other RNA viruses, e.g. other Paramyxoviruses are suitable.
  • the heterologous RNA sequence encodes an antigen of another mammalian pathogen, which is able to elicit a protective immune response, whereby the antigen is expressed by the BRSV mutant according to the invention upon replication in the host cell.
  • the heterologous gene is selected from the group of cattle pathogens, for example, Bovine Rotavirus, Bovine Viral Diarrhoea virus, Parainfluenza type 3 virus, , Bovine Herpesvirus, Foot and Mouth Disease virus and Pasteurella haemolytica.
  • cattle pathogens for example, Bovine Rotavirus, Bovine Viral Diarrhoea virus, Parainfluenza type 3 virus, , Bovine Herpesvirus, Foot and Mouth Disease virus and Pasteurella haemolytica.
  • heterologous RNA sequence may encode a cytokine.
  • cytokines e.g. interferons are known to play an important role as immune modulators.
  • a virus mutant according to the present invention and in particular a live BRSV, optionally expressing one or more different heterologous polypeptides of specific pathogens can, when it has an attenuated character, be used to vaccinate mammals.
  • Vaccination with such a live vaccine or live vector vaccine is followed by replication of the BRSV mutant within the inoculated host, expressing in vivo the BRSV polypeptides, along with heterologous polypeptides if the encoding genes are inserted.
  • the polypeptides expressed in the inoculated host will then elicit an immune response against both BRSV and the specific pathogen.
  • heterologous polypeptide derived from the specific pathogen can stimulate a protective immune response, then the mammal inoculated with the BRSV mutant according to the invention will be immune to subsequent infection by that pathogen as well as to infection by BRSV.
  • a heterologous nucleic acid sequence incorporated into the region of F-gene where the IFCSP has been deleted in the RSV according to the invention may be expressed in vivo during several replication cycles, providing a solid, safe and long-lasting immunity to the pathogen from which it was derived.
  • Another embodiment of the invention relates to vaccines for the protection of mammals against Bovine Respiratory Syncytial virus infection.
  • vaccines comprise a Bovine Respiratory Syncytial virus according to the invention and a pharmaceutically acceptable carrier.
  • a mutant virus according to the invention containing and expressing one or more different heterologous polypeptides can serve as a multivalent vaccine.
  • the BRSV mutant according to the present invention can be grown on susceptible cells, e.g. on a cell culture of bovine origin.
  • the viruses thus grown can be harvested by collecting the tissue cell culture fluids and/or cells.
  • the live vaccine may be prepared in the form of a suspension or may be lyophilized.
  • viruses according to the invention may also be inactivated and, for example, be used in a vaccine in inactivated form together with a suitable adjuvant.
  • the vaccine comprises a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carriers or diluents include e.g. materials as simple as sterile water or physiological salt solution.
  • stabilizers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk, plant hydrolysates and buffers (e.g. phosphate buffer).
  • one or more compounds having adjuvant activity may be added to the vaccine.
  • Suitable adjuvants are for example aluminum hydroxide, phosphate or oxide, oil-emulsions e.g. of Bayol F(R) or Marcol 52(R), saponins or vitamin-E solubilisate. These adjuvants have the advantage that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to the vaccine.
  • the vaccine comprises an adjuvant.
  • the useful effective amount to be administered will vary depending on the age and weight of the animal, the mode of administration and type of pathogen against which vaccination is sought. Nevertheless, since the live attenuated viruses according to the invention are self- propagating, the amount of virus initially administered is not critical.
  • a suitable dosage can be for example about 10. 3 - 0 - 10. 7 - 0 pfu/mammal.
  • the virus mutant according to the invention can be given inter alia intranasally, intradermally, subcutaneously or intramuscularly.
  • the vaccine according to the present invention is in a freeze-dried form.
  • Another embodiment of the present invention relates to methods for the preparation of a vaccine according to the invention.
  • such methods comprise admixing a virus according to the invention and a pharmaceutically acceptable carrier or diluent.
  • methods may comprise admixing adjuvants, freeze-drying and other preparations known in the art.
  • Figure 1 Construction of mutagenized BRSV F ORFs
  • Figure 2 Plaque sizes of recombinant BRSV
  • Figure 3 Growth curves of recombinant BRSVs
  • Figure 4 Penetration kinetics of recombinant BRSVs
  • Figure 5 Insertion of arbitrary choosen amino acid sequence and second furin cleavage site into gB of BHV-1.
  • Figure 6 Presence of the modified gB ORF in recombinant BHV-1/gBFu2.
  • Figure 7 Processing of gBFu2.
  • the F ORFs were isolated after cleavage of the respective pspF plasmids with Ncol and
  • BSR T7/5 cells (U. Buchholz et al. J. Virol) stably expressing T7 RNA polymerase were grown overnight to 80% confluency in 32 mm-diameter dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Cells were transfected with a plasmid mixture containing 10 ⁇ g of full length plasmid (p rBRSV), 4 ⁇ g of pN, 2 ⁇ g of pM2, and 2 ⁇ g of pL.
  • Transfection experiments were carried out with a mammalian transfection kit (CaPO 4 transfection protocol, Stratagene). Five days after transfection cells were split at a ratio of 1:3.
  • MDBK Madin-Darby bovine kidney
  • MDBK cells were infected with approximately 100 plaque forming units (pfu) and overlaid with semi-solid medium containing 0.75% methylcellulose. After a 6 days incubation the cultures were fixed with 3% paraformaldehyde in phosphate buffered saline without Mg 2+ and Ca 2+ (PBS) and 0.2% Triton X-100.
  • Plaque diameters were measured microscopically and indicated as mean values of at least 50 plaques.
  • MDBK cells were infected with rBRSV at a multiplicity of infectivity (moi) of 0.04. After 2h of incubation at 37°C inocula were removed, the cultures were rinsed twice with PBS, and supplemented with cell culture medium. At the times indicated the supernatants were collected respectively the entire cultures were frozen at -70 °C. Infectivity was determined by titration of serial dilutions on MDBK cells . Plaques were counted after a 6 days incubation under semi-solid medium overlay. 3. Penetration kinetics
  • Prechilled MDBK cells were incubated with approximately 300 pfu of rBRSV, Fins-F BRSV, for 2h at 4 °C.
  • Non-adherent virus was removed by thoroughly rinsing with icecold PBS. After shifting the incubation temperature from 4 °C to 37 °C penetration was terminated at the times indicated.
  • Cell-associated extracellular virus was inactivated through low pH with citric buffer (40 mM citric acid, 10 mM KCI, 135 mM NaCI, pH 3.0). Plaques were counted after 6d of incubation under semi-solid medium. The plaque counts of untreated control cultures were defined as 100% penetration. 4.
  • Bovine pharyngeal cells (KOP-R) were grown overnight in 3,2 cm-diameter plastic dishes to 80% confluence. Transient transfection experiments were performed with 2 ⁇ g of the BRSV F expression plasmids with 0,5 ⁇ g of the transactivator plasmid pAMB 25, encoding the 89 K MCMVie phosphoprotein (Koszinowski etal., J.Virol, 58, 59-66,1986). Superfect® transfection reagent was added according to the manufacturer's protocol (Qiagen, Hilden, Germany). After 24 h the transfection medium was replaced by cell culture medium.
  • FCS2 furin cleavage site
  • the gBFu2 ORF was used to rescue a gB-negative mutant of the laboratory strain BHV-1 /Horst, by cotransfecting purified viral DNA and plasmid pgBFu2 DNA into bovine pharyngeal cell line KOP/R.
  • a single plaque isolate from the infectious progeny virus (referred to as BHV-1/gBFu2) was used for further characterization.
  • plasmid was named pgB/Fu2.
  • PCR and cloning were done according to established procedures (Fritsch, Sambrook etc.).
  • PCR oligonucleotides gbfu2-1 +: gaggcgccaagcgcgaggcgatagtcaaggctgactctagagagctcaag gbfu2-2+: taagaattcgggcccgcgacgtgcgcgccgaggcgccaagcgagcggcg gbfu2-1 -: cgccgggcgcagacggcgcggcgcggcgcttgcgcttgagctctctagag gbfu2.2-: agtaagcttgggcccgttggccgcgcggtccgcgggcgcgggcgcggcgcgggcgcgggcgcgggcgcggcgcgggcgcgggcgcgggcgcggcgcgggcgcggcggg
  • KOP-R cells were cotransfected with 1 ⁇ g purified gB-negative BHV-1 DNA and 5 ⁇ g DNA of pgB/Fu2. Infectious progeny from the transfection was titrated. Virus from single plaques were plaque purified and one isolate named BHV-1 /gBFu2 was further characterized by Southern blotting of purified viral DNA cleaved with BamHI and BamHI/Xbal using a 32 P- labelled probe from the BHV-1 gB-gene.

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Abstract

La présente invention se rapporte à des virus contenant des glycoprotéines ayant des sites de liaison. Pour BRSV, on a découvert que 27AA IFCSP ne joue pas un rôle essentiel dans l'activité de fusion cellulaire de la protéine F et que la suppression ou le remplacement d'au moins une partie de IFCSP ne détruit pas la fonction F dans le cadre d'une réplication BRSV productive. Le retrait de Gly110 à Arg136 et l'expression transitoire de la protéine obtenue (Fins) se sont traduits par la formation de bourgeons syncytiaux s'avérant légèrement plus petits que ceux induits par wt F. Par conséquent, on a découvert que les oligopeptides ou les protéines bioactives peuvent être intégré(e)s entre deux sites de clivage par la furine d'une glycoprotéine de support, notamment les sites de clivage par la furine d'une protéine F du RSV. En raison du traitement de la protéine précurseur, se traduisant en clivage au niveau des sites de clivage par la furine, la séquence hétérologue peut être excisée et, en conséquence, sécrétée par une cellule infectée par le virus. Cette approche est également valable pour d'autres glycoprotéines contenant deux sites de clivage par la furine, dans lesquels la séquence originale entre les sites de clivage peut être (partiellement) remplacée par une séquence hétérologue ou simplement supprimée. Dans des protéines avec un site de clivage par la furine, les séquences hétérologues peuvent être insérées avec une séquence pour un second site de clivage, ce qui a pour effet de créer une situation dans laquelle la séquence hétérologue est à nouveau flanquée de deux sites de clivage par la furine dans la protéine recombinante. Le traitement de la protéine mutée se traduit également par l'excision de la séquence hétérologue susceptible d'être sécrétée par les cellules infectées par le virus. On peut utiliser les virus, dans lesquels les glycoprotéines sont modifiées selon une des manières susmentionnée, à des fins vaccinales.
PCT/EP2003/002520 2002-04-03 2003-03-12 Mutants viraux manipules dans les sites de clivage par la furine de glycoproteines WO2003083095A1 (fr)

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EP1622928A2 (fr) * 2003-05-09 2006-02-08 Research Development Foundation Insertion de sites de clivage de furine protease dans des proteines de membrane et leurs utilisations
WO2006063445A1 (fr) * 2004-12-14 2006-06-22 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Vaccin recombine contre la fievre aphteuse
WO2011008974A3 (fr) * 2009-07-15 2011-04-28 Novartis Ag Compositions à base de protéine f du vrs et procédés de fabrication associés

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1622928A4 (fr) * 2003-05-09 2006-06-14 Res Dev Foundation Insertion de sites de clivage de furine protease dans des proteines de membrane et leurs utilisations
US7521209B2 (en) 2003-05-09 2009-04-21 Research Development Foundation Insertion of furin protease cleavage sites in membrane proteins and uses thereof
EP1622928A2 (fr) * 2003-05-09 2006-02-08 Research Development Foundation Insertion de sites de clivage de furine protease dans des proteines de membrane et leurs utilisations
US8409588B2 (en) 2004-12-14 2013-04-02 Markus Czub Recombinant foot and mouth disease vaccine
WO2006063445A1 (fr) * 2004-12-14 2006-06-22 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Vaccin recombine contre la fievre aphteuse
EP3988115A3 (fr) * 2009-07-15 2022-08-17 GlaxoSmithKline Biologicals S.A. Compositions à base de protéine f du vrs et procédés de fabrication associés
EP3178490A3 (fr) * 2009-07-15 2017-08-30 GlaxoSmithKline Biologicals S.A. Compositions à base de protéine f du vrs et procédés de fabrication associés
US11261239B2 (en) 2009-07-15 2022-03-01 Glaxosmithkline Biologicals Sa RSV F protein compositions and method for making same
WO2011008974A3 (fr) * 2009-07-15 2011-04-28 Novartis Ag Compositions à base de protéine f du vrs et procédés de fabrication associés
US11629181B2 (en) 2009-07-15 2023-04-18 Glaxosmithkline Biologicals Sa RSV F protein compositions and methods for making same
US11655284B2 (en) 2009-07-15 2023-05-23 Glaxosmithkline Biologicals Sa RSV F protein compositions and methods for making same
EP4183412A1 (fr) * 2009-07-15 2023-05-24 GlaxoSmithKline Biologicals S.A. Compositions à base de protéine f du vrs et procédés de fabrication associés
EP4218799A1 (fr) * 2009-07-15 2023-08-02 GlaxoSmithKline Biologicals S.A. Compositions de protéine f rsv et leurs procédés de fabrication
EP4218800A1 (fr) * 2009-07-15 2023-08-02 GlaxoSmithKline Biologicals S.A. Compositions de protéine f rsv et leurs procédés de fabrication
US11820812B2 (en) 2009-07-15 2023-11-21 Glaxosmithkline Biologicals Sa RSV F protein compositions and methods for making same
US11827694B2 (en) 2009-07-15 2023-11-28 Glaxosmithkline Biologicals Sa RSV F protein compositions and methods for making same

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