WO2001021203A9 - Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale - Google Patents
Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasaleInfo
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- WO2001021203A9 WO2001021203A9 PCT/FR2000/002626 FR0002626W WO0121203A9 WO 2001021203 A9 WO2001021203 A9 WO 2001021203A9 FR 0002626 W FR0002626 W FR 0002626W WO 0121203 A9 WO0121203 A9 WO 0121203A9
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- protein
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- immunogenic peptide
- ompa
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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
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- 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
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- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
- A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
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- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6068—Other bacterial proteins, e.g. OMP
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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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
- 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
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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
- 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
- the invention relates to the use of an enterobacterium membrane protein OmpA, in particular the protein P40 of Klebsiella pneumoniae, associated with an immunogenic peptide derived from respiratory syncytial virus (RSV) for the preparation of a pharmaceutical composition which can be administered by the nasal spray intended to induce a protective immune response of the upper respiratory tract and the lower respiratory tract (lungs) of a subject against infection by RSV.
- OmpA enterobacterium membrane protein
- RSV respiratory syncytial virus
- the invention includes the use of these compounds for the preparation of a vaccine for the prevention and treatment of RSV infection.
- RSV is the most common cause of hospitalization of infants under one year of age for acute respiratory infections. Children with laryngotracheobronchitis, bronchiolitis and pneumonia require hospital care and in infants with congenital heart disease, the death rate is over 37%. Other disorders such as bronchopulmonary dysplasia, kidney disease and immunodeficiency are all factors responsible for high mortality. RSV infections can also be a cause of death in the elderly.
- RSV epidemic occurs during the winter period from November to April and the greatest incidence of serious illness occurs in infants 2 to 6 months of age.
- RSV-A and RSV-B by the antigenic variation of the glycoprotein G of RSV: subgroup A and subgroup B, which circulate concurrently.
- subgroup A and subgroup B which circulate concurrently.
- a recent study in France from 1982 to 1990 showed an alternation from one subgroup to another over a period of 5 years.
- Strain A is often the cause of more serious infections than strain B.
- Human RSV belongs to the genus pneumovirus, a member of the family of
- the virus genome consists of a strand of RNA with negative polarity, not segmented, coding for 10 distinct proteins: NS1, NS2, N; P, M, SH (or IA), G, F, M2 (or 22K) and L (Murphy et al., Virus Res. 32: 13-36, 1994).
- the fusion glycoprotein F synthesized as a precursor FO is split into two subunits F1 (48 kDa) and F2 (20 kDa) linked by disulfide bridges. Protein F is conserved between VRS-A and VRS-B (91% homology). Conversely, the attachment glycoprotein G is very variable from one subgroup to another. Only one region of 13 amino acids (aa164-aa176) is highly conserved and in particular the position of four cysteine residues (173, 176, 182 and 186) is kept constant in all human and bovine RSVs. It has been shown in animal models that the two glycoproteins F and G play a major role in protection against RSV.
- the monoclonal antibodies directed against G and F are capable of neutralizing the virus in vitro and passively administered, they protect the cotton rat against infection by RSV.
- conventional vaccines that is, formalin-inactivated RSV, analogous to measles and measles vaccines, failed. Instead of providing protection in vaccinated children, this type of vaccine has the effect of potentiating natural viral disease.
- Current treatments for worsening RSV disease in infants are airway congestion relief by aspiration of phlegm and respiratory assistance by ventilation.
- An antiviral, Ribavirin seems to be effective in severely affected cases. However, its use in pediatric therapy is still poorly defined.
- Passive immunization with anti-RSV immunoglobulins is an alternative route in the treatment of serious RSV infections: no undesirable side effects have been observed. However, this type of treatment is very expensive and difficult to extrapolate on a large scale.
- the present invention relates to the use of an enterobacterium protein OmpA, or one of its fragments, associated with an immunogenic peptide derived from the respiratory syncytial virus (RSV) for the preparation of an administrable pharmaceutical composition.
- RSV respiratory syncytial virus
- said induced immune response protects both the upper respiratory tract and the lower respiratory tract (lungs) of a subject against infection by RSV.
- protein will also be understood to denote the peptides or polypeptides and the term “OmpA” (for “Outer Membrane Protein”), the proteins of the outer membrane of type A.
- fragment of an OmpA protein is intended to denote in particular any fragment of the amino acid sequence included in the amino acid sequence of the OmpA protein which, when associated with an RSV immunogenic peptide, optionally in the presence an immunity adjuvant, is capable of generating or increasing an antibody-type immune response directed against said immunogenic peptide, said fragment of the OmpA protein comprising at least 10 consecutive amino acids, preferably at least 15 consecutive amino acids or more preferably at least 20 consecutive amino acids of the amino acid sequence of said OmpA protein.
- immunogenic peptide derived from the respiratory syncytial virus is intended to denote in particular any peptide whose amino acid sequence comprises at least 5 consecutive amino acids, preferably at least 10 or 15 consecutive amino acids of the sequence of a protein. expressed by the VRS, said peptide comprising in its sequence an antigenic determinant or epitope capable of inducing an antibody-type immune response (dependent B lymphocytes) exerted against said “B” epitope, in particular in the presence of said OmpA protein or one of its fragments, in an organism previously immunized with said immunogenic peptide
- the invention comprises the use according to the invention, characterized in that said enterobacterium is Klebsiella pneumomae.
- a subject of the invention is also the use according to the invention, characterized in that the amino acid sequence of said OmpA protein, or one of its fragments, comprises a) the amino acid sequence of sequence SEQ ID No. 2, b) the amino acid sequence of a sequence having a percentage identity of at least 80%, preferably 90%, 95% or 97%, after optimal alignment with the sequence SEQ ID N ° 2, or c) the amino acid sequence of a fragment of at least 10 consecutive amino acids of the sequence SEQ ID No. 2.
- percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.
- Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window” to identify and compare the regions. local sequence magnetization.
- the optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the algorithm of local homology of Smith and Waterman (1981) [Ad. App. Math.
- the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences by "comparison window" in which the region of the sequence of nucleic acid or amino acids to be compared can include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences.
- the percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
- the invention comprises the use of an enterobacterium OmpA protein or of a fragment thereof according to the invention, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained by an extraction process from a culture of said enterobacterium.
- the invention also comprises the use of an enterobacterium OmpA protein or of a fragment thereof according to the invention, characterized in that said enterobacterium OmpA protein, or the one of its fragments, or said immunogenic peptide is obtained recombinantly.
- the invention relates to the use according to the invention, characterized in that said immunogenic peptide is chosen from peptides derived from protein G, F, M2 or N of RSV, preferably protein G.
- said immunogenic peptide is a fragment of protein G comprising a type B epitope.
- type B epitope included in a fragment of the G protein of RSV is intended to denote in particular an amino acid sequence of at least 5, 10 or 15 consecutive amino acids of a fragment of the G protein capable to induce an antibody-type immune response (dependent B lymphocytes) directed against said amino acid sequence, in particular in the presence of the OmpA P40 protein of sequence SEQ ID No. 1.
- said immunogenic peptide is chosen from the peptides of sequence SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 9 or SEQ ID No 11, or of amino acid sequence having an identity percentage of at least 80%, preferably 90%, 95% or 97%, after optimal alignment with the sequence SEQ ID N ° 5, SEQ ID N ° 6 , SEQ ID N ° 7, SEQ ID N ° 9 or SEQ ID N ° 11.
- the present invention also relates to the use according to the invention, characterized in that said immunogenic peptide is coupled or mixed with said enterobacterium protein OmpA, or one of its fragments.
- the invention relates to the use according to the invention, characterized in that the pharmaceutical composition comprises a nucleic construct coding for said enterobacterium protein OmpA, or one of its fragments, and / or a nucleic construct coding for said immunogenic peptide, said nucleic construct being contained in an expression vector or in a transformed host cell capable of expressing said enterobacterium OmpA protein, or one of its fragments, and / or said peptide immunogenic.
- the invention also includes the use according to the invention, characterized in that said immunogenic peptide is coupled by covalent bond, in particular by chemical coupling, with said OmpA protein or one of its fragments, to form a hybrid protein.
- the use according to the invention is characterized in that one or more binding elements are introduced into said OmpA protein, or one of its fragments, and / or into said immunogenic peptide for facilitate chemical coupling, preferably, said connecting element introduced is an amino acid.
- the invention it is possible to introduce one or more binding elements, in particular amino acids to facilitate the coupling reactions between the OmpA protein, or one of its fragments, and said immunogenic peptide.
- the covalent coupling between the OmpA protein, or one of its fragments, and said immunogenic peptide according to the invention can be carried out at the N- or C-terminal end of the OmpA protein, or one of its fragments.
- the bifunctional reagents allowing this coupling will be determined as a function of the end of the OmpA protein, or one of its fragments, chosen to effect the coupling and of the nature of said immunogenic peptide to be coupled.
- the use according to the invention is characterized in that the hybrid protein formed by covalent coupling between said immunogenic peptide and said OmpA protein, or one of its fragments, is an expressed fusion protein by a cell transformed with a nucleic construct coding for said hybrid protein.
- the hybrid proteins resulting from covalent coupling between said immunogenic peptide and said OmpA protein, or one of its fragments can be prepared by genetic recombination.
- the hybrid or chimeric protein can be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for said OmpA protein, or one of its fragments, of a sequence coding for said immunogenic peptide.
- the methods for synthesizing hybrid molecules include the methods used in genetic engineering to construct hybrid polynucleotides encoding the desired polypeptide sequences.
- the invention is characterized in that said enterobacterium protein OmpA, or one of its fragments, and said immunogenic peptide derived from the respiratory syncytial virus used for the preparation of a pharmaceutical composition administered by nasal route according to the invention. are in the form of a hybrid protein obtained by covalent coupling, by chemical synthesis or by genetic fusion, between the p40 protein of Klebsiella pneumoniae of sequence SEQ ID No.
- amino acid sequence having a percentage identity of at least 80%, preferably 90%, 95% or 97% after optimal alignment with the sequence SEQ ID No 2, and the immunogenic peptide derived from the RSV of sequence SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 9, SEQ ID No 11, or sequence amino acids with an identity percentage of at least 80%, preferably 90%, 95% or 97%, after optimal alignment with the sequence SEQ ID N ° 5, SEQ ID N ° 6, SEQ ID N ° 7, SEQ ID N ° 9 ⁇ or SEQ ID N ° 11.
- the invention also relates to the use according to the invention, characterized in that the pharmaceutical composition comprises a nucleic construct coding for said hybrid protein, in particular contained in an expression vector, or in a host cell transform containing said nucleic construct capable of expressing said hybrid protein.
- OmpA in particular P40 from K. pneumoniae, associated in particular by covalent bond to an immunogenic peptide derived from RSV, has the surprising property of potentiating the immune response for some of these immunogenic peptides without it being necessary to resort to the addition of an adjuvant.
- said pharmaceutical composition will optionally comprise an immunity adjuvant.
- the invention also includes the use according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity, in particular encapsulated in a liposome.
- the invention comprises the use according to the invention, characterized in that said vehicle is a viral vector containing a nucleic construct coding for said protein OmpA, or one of its fragments, for said immunogenic peptide, or for said hybrid protein, or a transformed host cell containing said nucleic construct capable of expressing said OmpA protein, or one of its fragments, said immunogenic peptide, or said hybrid protein.
- said vehicle is a viral vector containing a nucleic construct coding for said protein OmpA, or one of its fragments, for said immunogenic peptide, or for said hybrid protein, or a transformed host cell containing said nucleic construct capable of expressing said OmpA protein, or one of its fragments, said immunogenic peptide, or said hybrid protein.
- said transformed host cell is a bacterium which is not pathogenic for mammals and said nucleic construct which it contains further comprises the elements necessary for the secretion or the expression on the surface of the membrane of said bacteria of said protein. OmpA, or a fragment thereof, of said immunogenic peptide, or of said hybrid protein.
- the present invention relates to the use according to the invention for the preparation of a pharmaceutical composition, in particular a vaccine composition, intended for preventing or treating infections with RSV.
- a pharmaceutical composition in particular a vaccine composition, intended for preventing or treating infections with RSV.
- the examples which follow are intended to illustrate the invention without in any way limiting its scope.
- Figure 1 SDS PAGE 12% gel under denaturing conditions of the recombinant P40 protein (rP40) of sequence SEQ ID No. 2 (37 Kda), molecular size markers (M) in decreasing order (200, 116, 97.4, 66, 45, 31, 21.5 Kda), and of the protein P40G2Na of sequence SEQ ID No. 4 (49 Kda).
- rP40 recombinant P40 protein
- M molecular size markers
- Figures 2A to 2C Evaluation of the systemic IgG response (Figure 2A) and isotyping of serum IgG in naive mice ( Figure 2B) or presensitized with Klebsiella pneumoniae (Figure 2C), having received 3 administrations intra-nasally (in) 40 ⁇ g of G2Na protein of sequence SEQ ID No. 9 fused to rP40.
- Figures 3A and 3B Immunogenicity of the P40-G5 conjugates, administered i.n. to BABL / c mice. 10 ⁇ g of G5 equivalents coupled to rP40 are administered 3 times, 10 days apart, to naive (FIG. 3A) or presensitized (FIG.
- FIG. 3B mice by Klebsiella pneumoniae. 10 days after the last immunization, the animals are punctured and the serum IgG directed against G5 assayed by ELISA.
- Figures 4A and 4B Study of the protection of the upper ( Figure 4A) and lower ( Figure 4B) pathways after immunization of BALB / c mice with the fusion protein P40G2Na.
- FIGS. 5A and 5B Study of the protection of the lower (FIG. 5A) and upper (FIG. 5B) pathways after immunization of BABL / c mice with the P40-G5 conjugate.
- the DNA encoding the P40 protein was obtained by PCR amplification from the genomic DNA of Klebsiella pneumoniae IP 1145 (Nguyen et al., Gene 210: 93-101, 1998).
- the gene fragment coding for P40 is inserted into various expression vectors, in particular a vector under the control of the promoter of the Trp operon: pvaLP40 (5.7 Kpb).
- the nucleotide sequence and the peptide sequence of the P40 protein are represented respectively by the sequences SEQ ID No. 1 and SEQ ID No. 2.
- a producing strain £. coli K12 was transformed with an expression vector pvaLP40 (5.7 Kbp).
- the rP40 protein is produced under form of inclusion body with a high yield (> 10%, g of protein / g of dry biomass). This example is only an illustration of the expression of the rP40 protein, but it can be extended to other bacterial strains as well as other expression vectors.
- EXAMPLE 2 Cloning of the DNA Coding for the P40G2Na Protein
- the DNA coding for the G2Na fragment of sequence SEQ ID No. 9 of the protein G of VRS-A was obtained by assembling synthetic oligonucleotides on solid supports (Dynal magnetic beads, Oslo) as described by Nguyen et al. (M. Uhlen, E. Homes, and O. OIsvik (eds.), Advances in biomagnetic separation. Eaton Publishing Co., Natick, p. 73-78, 1994), the oligonucleotides were designed and optimized with the usual codon from Escherichia coli.
- the G2Na DNA was cloned into the vector pvaLP40 described in Example 1, the resulting expression vector is named pvaLP40G2Na (6 Kpb).
- the nucleotide sequence and the peptide sequence of P40G2Na are represented respectively by the sequences SEQ ID No. 3 and SEQ ID No. 4.
- the description of the following fermentation process is applicable to the expression of the rP40 protein and that of the P40G2Na protein.
- the culture conditions are given by way of example and can be optimized to obtain a better yield of expressed proteins.
- the inoculation is carried out with the recombinant E. coli strain described above in an Erlenmeyer flask containing 250 ml of TSB medium (Tryptic Soy Broth, Difco) containing Ampicillin (100 ⁇ g / ml, Sigma) and Tetracyciine ( 8 ⁇ g / ml, Sigma), which is incubated overnight at 37 ° C. Then 2 liters of culture medium in a fermenter (Biolafitte, France) are seeded using 200 ml of the culture previously obtained.
- TSB medium Troptic Soy Broth, Difco
- Ampicillin 100 ⁇ g / ml, Sigma
- Tetracyciine 8 ⁇ g / ml, Sigma
- the culture medium can be composed of chemical agents, supplemented with vitamins, yeast extracts, known to have a high density growth of bacterial cells, as described by Nguyen et al. (Gene 210: 93-101, 1998).
- the parameters controlled during fermentation are: pH, agitation, temperature, oxygenation rate, feeding of combined sources (Glycerol or Glucose).
- the pH is regulated at 7.0, the temperature is fixed at 37 ° C.
- Growth is controlled by supplying glycerol (87%) at a constant flow rate (12 ml / h) to maintain the voltage signal for dissolved oxygen at 30%.
- the cells After centrifuging the culture broth (4000 rpm, 10 min, 4 ° C), the cells are resuspended in a 25 mM Tris-HCl buffer pH 8.5.
- the insolubles or inclusion bodies are obtained after treatment with lysozyme (0.5 g / liter, 1 hour at room temperature / gentle stirring).
- the inclusion body pellet obtained by centrifugation (15 min at 10,000 g at 4 ° C) is taken up in 25 mM Tris-HCl buffer at pH 8.5 and 5 mM MgCl 2 , then centrifuged (15 min at 10 000 g).
- the inclusion bodies at 37 ° C. are dissolved for 2 hours in a buffer
- the dialysate is deposited on a column containing a support of the strong anion exchanger type (Biorad Macro Prep High Q gel) balanced in the buffer described above at a linear flow rate of 15 cm / h. Proteins are detected at 280 nm.
- the protein rP40 is eluted, with a linear flow rate of 60 cm / h, for a concentration of 0.2 M in NaCl in the 25 mM Tris-HCl buffer pH 8.5 at 0.1% Zwittergent 3-14.
- the fractions containing the rP40 protein are mixed and concentrated by ultrafiltration using an Amicon shaking cell system used with a Diaflo membrane type YM10 (cutoff threshold 10 kDa) for volumes of the order of 100 ml, or using a Minitan Millipore tangential flow filtration system used with membrane plates having a cutoff threshold of 10 kDa for higher volumes.
- the fraction thus concentrated is dialyzed overnight at 4 ° C. against a 20 mM citrate buffer pH 3.0 containing 0.1% Zwittergent 3-14.
- the dialysate is deposited on a column containing a support of the strong cation exchanger type (Biorad Macro Prep High S gel) balanced in the 20 mM citrate buffer pH 3.0 containing 0.1% of Zwittergent 3-14.
- the rP40 protein is eluted (speed 61 cm / h) for a 0J M NaCl concentration.
- the bacteria cells After centrifuging the culture broth (4000 rpm, 10 min, 4 ° C), the bacteria cells are resuspended in a 50 mM Tris-HCl buffer pH 8.5 containing 1 mM EDTA, 0.2 M NaCl and 0 , 05% Tween 20.
- the insolubles or inclusion bodies are obtained after treatment with lysozyme (0.5 g / liter, 1 hour at room temperature / gentle stirring).
- the inclusion body pellet obtained by centrifugation (15 min at 10,000 g at 4 ° C) is taken up in a 25 mM Tris-HCl buffer at pH 8.5 containing 5 mM MgCl2, then centrifuged (15 min at 10,000 g).
- the inclusion bodies are dissolved at 37 ° C for 2 hours in a buffer
- the supernatant is then resuspended in 13 volumes of 25 mM Tris-HCl buffer pH 8.5 containing NaCl (8.76 g / 1) and Zwittergent 3-14 (0.1%, w / v). The solution is left overnight at room temperature with gentle stirring in contact with air.
- the dialysate is deposited on a column containing a support of strong anion exchanger type (Amersham Pharmacia Biotech Source Q gel) equilibrated in the buffer described above, and the P40G2Na protein is eluted using a NaCl gradient. in the same buffer. Proteins are detected at 280 nm.
- the fractions containing the P40G2Na protein are mixed and concentrated by ultrafiltration using an Amicon shaking cell system used with a Diaflo membrane of the YM10 type (cutoff threshold 10 kDa) for volumes of the order of 100 ml. , or using a Minitan Millipore tangential flow filtration system used with membrane plates having a cutoff threshold of 10 kDa for higher volumes.
- the fraction thus concentrated is dialyzed overnight at 4 ° C. against a 20 mM Tris-HCl buffer, pH 8.0, containing 0% Zwittergent 3-14.
- the dialysate is deposited on a column containing a support of the strong cation exchanger type (Amersham Pharmacia Biotech Source S gel) equilibrated in the preceding buffer, and the P40G2Na protein is eluted using an NaCl gradient in the same buffer. .
- the fractions of interest are mixed before being concentrated by ultrafiltration using one of the systems described above.
- Example 6 Chemical coupling to obtain P40-G5 A) Synthesis and characterization of the P40-G5Cvsa conjugate - Synthesis of the peptides G ⁇ aCys and CysG ⁇ a
- the peptide G5a represented by the sequence SEQ ID No. 5, is a peptide of 16 amino acids which corresponds to the aa144-aa159 fragment of protein G of VRS-A. It is obtained by chemical synthesis on solid phase starting from the C side terminai towards the N-terminal side.
- the peptides CysG ⁇ a and G5aCys represented respectively by the sequences SEQ ID No. 6 and SEQ ID No. 7, correspond respectively to this peptide with an additional Cysteine on the N- or C-terminal side which allows unequivocal coupling on a carrier protein.
- the peptides were synthesized using an automatic solid phase peptide synthesizer from the C side to the N-terminal side (FMOC chemistry on the scale of 0.1; 0.25 or 1.0 mmol).
- the synthesis of the CysG ⁇ a peptide is carried out from a Proline preloaded on a resin of HMP type, which allows after cleavage to obtain a free acid function on the C-terminal side or a resin of type Rink amide MHBA, which allows after cleavage to obtain an amide function on the C-terminal side. That of the G ⁇ aCys peptide begins with a Cysteine preloaded on one or the other of the resins.
- a coupling cycle takes place as follows: deprotection of the N-terminal amino function of the first amino acid using piperidine, activation of the acid function of the second amino acid to be coupled using HBTU / HMP and coupling.
- the peptide is cleaved from the resin and the side chains are deprotected by reaction with a water / TFA mixture.
- the peptide is precipitated with ether cooled beforehand to -40 ° C and the mixture is centrifuged. The pellet is washed three times with ether and then dried with nitrogen. The pellet is taken up with water containing 0.1% TFA. The suspension is again centrifuged and the supernatant which contains the peptide is separated from the pellet, which contains the resin.
- the crude peptide is purified by semi-preparative reverse phase HPLC. The homogeneity of the purified peptide is verified by reverse phase HPLC and by capillary electrophoresis (FZCE). The theoretical structure is confirmed by comparison of the compatibility of the mass measured by ES-MS type mass spectrometry, with the mass calculated from the theoretical amino acid sequence.
- Mass of purified peptide after lyophilization 50 mg (7 ⁇ % net amount of peptide or 68% yield).
- AA or aa amino acid
- Boc Tert Butoxycarbonyl
- BHA Bromo hydrosuccimidyl acid
- CE Capillary Electrophoresis
- ES-MS Electrospray - Mass Spectrometry
- FMOC Fluorenylmethoxycarbonyl
- FZCE Free Zone Capillary Electrophoresis
- HBTU 2- (1 H-Benzotriazole-1-yl) -1J, 3,3-tretramethyluroniumhexafluorophosphate
- HMP p-hydroxymethylphenoxymethyl polystyrene
- MBHA methylbenzhydrylamine
- NMP N-methyl-2-pyrrolidone
- Pmc 2,2,5J, 8-Pentamethylchroman-6-sulfonyl
- RP-HPLC Reverse Phase - High Performance Liquid Chromatography
- tBOC t-butyloxycarbonyl
- tBu
- the method used for coupling has been described by Haeuw et al. (Eur. J. Biochem. 256: 446-464, 1998).
- the heterobifunctional reagent, N-hydroxysuccinimide bromoacetate or HBA, used allows activation of the Lysine residues of the protein by bromoacetylation, then coupling of the peptide via the thiol group carried by the Cysteine residue introduced in the C-terminal position of the peptide.
- the P40 protein is dialyzed against a 0.1 M sodium phosphate buffer pH 7.0 containing 0.1% Zwittergent 3-14 overnight at 4 ° C.
- the dialysis protein is then activated using the HBA reagent. This is added at a rate of 1.2 mg (in 10 ⁇ l of dimethylformamide) per mg of P40, then the whole is placed under stirring for 1 hour at room temperature and in the dark.
- the activated P40 is desalted by molecular sieving chromatography on Pharmacia Sephadex G25 support. Elution of the protein is carried out using a 0.1 M sodium phosphate buffer pH 7.0 containing 0.1% Zwittergent 3-14. The fractions containing the activated P40 are combined.
- the number of coupled peptides is determined by HPLC assay of the amino acids after acid hydrolysis and derivatization using PITC (PicoTag Waters method).
- PITC PieroTag Waters method
- the quantification of the carboxymethylcysteine residue released according to the method described by Kolodny & Robey makes it possible to precisely determine the number of moles of G ⁇ Cysa peptide fixed per mole of P40 protein. This value is generally between ⁇ and 10 G ⁇ Cysa / P40.
- FIG. 2A shows an elevated serum IgG titer against G2Na following administration of the fusion protein by the in route.
- This titer is significantly higher in animals presensitized by Klebsiella pneumoniae and the addition of CTB ( cholera toxin subunit B) to the fusion protein also improves the intensity of the response and its homogeneity.
- Isotyping of the serum IgG response (FIGS. 2B and 2C) highlights a majority IgG1 response in the absence of CTB with an increase in the IgG1 and IgG2a titers in mice sensitized with K. pneumoniae (FIG. 2C). The effect of presensitization on isotyping is no longer perceptible when CTB is added to the fusion protein.
- BALB / c mice are immunized with the P40G2Na hybrid protein and then infected i.n. with the RSV-A.
- FIG. 4A shows that the administration of the carrier protein rP40 alone, administered i.n. with or without CTB has no consequence on the infection of mice with RSV-A.
- administration of the fusion protein P40G2Na to mice not sensitized by Klebsiella pneumoniae leads to a reduction of the viral titer by 2 log 10 for ⁇ of the 6 mice.
- sensitizing mice with Klebsiella pneumoniae significantly improves the protection of the pulmonary tract.
- FIG. MontreA shows that the administration of the peptide G ⁇ coupled to rP40 by the nasal route causes a significant reduction in viremia (p ⁇ 0.0 ⁇ ) in the lungs of the mice against infection with RSV-A.
- FIG. MontreA shows that the administration of the peptide G ⁇ coupled to rP40 by the nasal route causes a significant reduction in viremia (p ⁇ 0.0 ⁇ ) in the lungs of the mice against infection with RSV-A.
- FIG. MontreA shows that the administration of the peptide G ⁇ coupled to rP40 by the nasal route causes a significant reduction in viremia (p ⁇ 0.0 ⁇ ) in the lungs of the mice against infection with RSV-A.
- FIG. MontreA shows that the administration of the peptide G ⁇ coupled to rP40 by the nasal route causes a significant reduction in viremia (p ⁇ 0.0 ⁇ ) in the lungs of the mice against infection with RSV-A.
- FIG. MontreA shows that the administration of the peptide G ⁇ coupled to
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Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR0014246-8A BR0014246A (pt) | 1999-09-23 | 2000-09-22 | Utilização de uma proteìna ompa de enterobactéria, ou um de seus fragmentos, associada a um peptìdeo imunogênico derivado do vìrus respiratório sincicial |
EP00964347A EP1218029A1 (fr) | 1999-09-23 | 2000-09-22 | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale |
CA002385404A CA2385404A1 (fr) | 1999-09-23 | 2000-09-22 | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale |
JP2001524627A JP2003528812A (ja) | 1999-09-23 | 2000-09-22 | Rsvの免疫原性ペプチドと組み合わせた腸内細菌ompa膜タンパク質の経鼻投与可能なワクチンの製造のための使用 |
AU75301/00A AU7530100A (en) | 1999-09-23 | 2000-09-22 | Use of an outer membrane protein a of an enterobacterium associated with a rsv immunogenic peptide for preparing vaccines for intranasal administration |
MXPA02003132A MXPA02003132A (es) | 1999-09-23 | 2000-09-22 | Uso de una proteina a de membrana externa (ompa) de enterobacteria combinada con un peptido inmunogenico rsv para preparar vacunas para administracion intranasal. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9911888A FR2798857B1 (fr) | 1999-09-23 | 1999-09-23 | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale |
FR99/11888 | 1999-09-23 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001021203A1 WO2001021203A1 (fr) | 2001-03-29 |
WO2001021203A9 true WO2001021203A9 (fr) | 2002-05-10 |
Family
ID=9550162
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2000/002626 WO2001021203A1 (fr) | 1999-09-23 | 2000-09-22 | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP1218029A1 (fr) |
JP (1) | JP2003528812A (fr) |
CN (1) | CN1379682A (fr) |
AU (1) | AU7530100A (fr) |
BR (1) | BR0014246A (fr) |
CA (1) | CA2385404A1 (fr) |
FR (1) | FR2798857B1 (fr) |
MX (1) | MXPA02003132A (fr) |
WO (1) | WO2001021203A1 (fr) |
ZA (1) | ZA200202175B (fr) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2827605B1 (fr) * | 2001-07-20 | 2004-07-16 | Pf Medicament | Nouveaux peptides derives de la proteine g du vrs et leur utilisation dans un vaccin |
FR2828106A1 (fr) * | 2001-08-02 | 2003-02-07 | Pf Medicament | Utilisation d'une omp d'enterobacterie de faible masse moleculaire comme porteur et/ou adjuvant |
EP2487243A3 (fr) * | 2005-01-07 | 2013-08-28 | Alnylam Pharmaceuticals Inc. | Modulation de l'ARNi du VRS et utilisations thérapeutiques correspondantes |
JP5646620B2 (ja) * | 2009-07-17 | 2014-12-24 | インダストリー アカデミック コーポレーション ファウンデーション, ハルリム ユニバーシティー | リポソームに被包されたオリゴヌクレオチド及びエピトープを含む免疫増強用組成物 |
KR20220119527A (ko) * | 2012-08-01 | 2022-08-29 | 버베리안 노딕 에이/에스 | 재조합 변형된 백시니아 바이러스 앙카라(ankara) (mva) 호흡기 신시티알 바이러스(rsv) 백신 |
CN103243112A (zh) * | 2013-05-16 | 2013-08-14 | 河北科星药业有限公司 | 预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1331355C (fr) * | 1986-04-21 | 1994-08-09 | Bioenterprises Pty. Ltd | Immunopotentialisation |
FR2726472B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Proteine porteuse a effet adjuvant, complexe immunogene la contenant, leur procede de preparation, sequence nucleotidique et vaccin |
FR2726577B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Procede d'obtention d'un peptide derive du virus respiratoire syncitial, polypeptide et bacterie l'exprimant, et leurs applications a titre de medicament |
FR2766192B1 (fr) * | 1997-07-17 | 2001-07-13 | Pf Medicament | Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie |
BR9815496A (pt) * | 1997-07-18 | 2001-12-18 | Connaught Lab | Vacinas de ácido nucléico que codificam proteìna gde vìrus sincicial respiratório |
FR2776521B1 (fr) * | 1998-03-27 | 2000-12-15 | Pf Medicament | Utilisation de conjugues p40 actifs par voie nasale |
-
1999
- 1999-09-23 FR FR9911888A patent/FR2798857B1/fr not_active Expired - Fee Related
-
2000
- 2000-09-22 AU AU75301/00A patent/AU7530100A/en not_active Abandoned
- 2000-09-22 WO PCT/FR2000/002626 patent/WO2001021203A1/fr not_active Application Discontinuation
- 2000-09-22 CA CA002385404A patent/CA2385404A1/fr not_active Abandoned
- 2000-09-22 MX MXPA02003132A patent/MXPA02003132A/es unknown
- 2000-09-22 BR BR0014246-8A patent/BR0014246A/pt not_active Application Discontinuation
- 2000-09-22 CN CN00814283A patent/CN1379682A/zh active Pending
- 2000-09-22 JP JP2001524627A patent/JP2003528812A/ja not_active Withdrawn
- 2000-09-22 EP EP00964347A patent/EP1218029A1/fr not_active Withdrawn
-
2002
- 2002-03-18 ZA ZA200202175A patent/ZA200202175B/xx unknown
Also Published As
Publication number | Publication date |
---|---|
ZA200202175B (en) | 2002-12-24 |
FR2798857B1 (fr) | 2003-06-06 |
CN1379682A (zh) | 2002-11-13 |
EP1218029A1 (fr) | 2002-07-03 |
AU7530100A (en) | 2001-04-24 |
MXPA02003132A (es) | 2002-09-30 |
WO2001021203A1 (fr) | 2001-03-29 |
FR2798857A1 (fr) | 2001-03-30 |
CA2385404A1 (fr) | 2001-03-29 |
JP2003528812A (ja) | 2003-09-30 |
BR0014246A (pt) | 2002-05-21 |
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