EP0789768A1 - Protein g von respiratory syncitial virus, exprimiert auf bakterie-membran - Google Patents
Protein g von respiratory syncitial virus, exprimiert auf bakterie-membranInfo
- Publication number
- EP0789768A1 EP0789768A1 EP95939337A EP95939337A EP0789768A1 EP 0789768 A1 EP0789768 A1 EP 0789768A1 EP 95939337 A EP95939337 A EP 95939337A EP 95939337 A EP95939337 A EP 95939337A EP 0789768 A1 EP0789768 A1 EP 0789768A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence
- protein
- bacterium
- polypeptide
- heterologous polypeptide
- 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- Withdrawn
Links
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Classifications
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/882—Staphylococcus
Definitions
- the present invention relates to a method of recombinant DNA technique making it possible to modify punctually by site-directed mutagenesis, nucleotides in a gene coding for a polypeptide sequence, useful in particular for obtaining vaccines by the oral route against the respiratory syncitial virus ( VRS); it also relates to recombinant peptides and bacteria capable of being obtained by this process, the compositions containing them, as well as the corresponding nucleotide sequences.
- VRS respiratory syncitial virus
- the respiratory syncytial virus (RSV) is the most common cause of respiratory diseases in newborns: bronchopneumopathies
- RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a non-segmented RNA genome, of negative polarity, coding for 10 specific proteins.
- RSV structural proteins for a vaccine such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a protein of 9.5 Kd, or the major capsid protein (protein N).
- Application WO 89/02935 describes the protective properties of the entire F protein of RSV, optionally modified in monomeric or desacelylated form.
- RSV infections of the upper airways treatment is essentially based on symptomatic medications identical to those of other viral infections.
- RSV infections of the lower airways treatment in infants is based on maintaining proper hydration, aspirating secretions and administering oxygen if necessary.
- a positive effect has been observed with ribavirin, a nucleotide active in vitro against
- the subject of the present invention is a process for obtaining a peptide or a protein, characterized in that a non-pathogenic bacterium for mammals is introduced: a) a DNA sequence coding for a heterologous polypeptide carried by a peptide sequence between amino acid residues 130 and 230 of protein G of the respiratory syncytial virus, or a peptide sequence having at least 80% homology with said peptide sequence, b) the means allowing the expression of the polypeptide on the surface of the membrane of the bacteria.
- the heterologous peptide can be expressed fused with a fragment allowing the anchoring in the membrane of the bacterium.
- Protein G is an RSV envelope glycoprotein, with a molecular weight between 84 and 90 Kd, poor in methionine.
- the sequence of protein G differs for subgroups A and B of RSV; the terms "protein G sequence" when used in the present application, should be understood as referring to both the sequence of subgroup A or of subgroup B, when this is not specified differently.
- the Applicant has demonstrated that the sequence between amino acids 130 and 230 of the natural protein G is particularly suitable for inducing effective protection against infection by RSV, subgroups A and B, without inducing the pathologies observed. with vaccines based on the whole virus inactivated by formalin, or observed with whole F and G proteins.
- the means allowing the expression of the polypeptide are known to those skilled in the art and are adapted according to the bacteria used.
- the DNA sequence is introduced in the form of a plasmid, such as a shuttle plasmid.
- the DNA sequence is integrated into the chromosome of the bacterium RSV proteins have to date been expressed in different expression systems such as vaccinia virus, baculoviruses or adenoviruses .
- potential problems are associated with the presence of residual virus particles.
- the method according to the present invention uses commensal bacteria from humans, apathogenic and edible.
- gram-positive bacteria are used, in particular the bacteria may belong to the genus Staphylococcus.
- Staphylococcus xylosus and Staphylococcus carnosus which are bacteria used in the food industry for many years, and can be administered, alive, orally.
- Expression systems of heterologous epitopes on the surface of S. xylosus have been described in particular by N'guyen et al in Gene, 1993, 128. 89-94, as well as for S. carnosus by Samuelsson et al in J. Bacteriol. , 1995, 177, 1470-1476.
- the heterologous polypeptide is expressed on the surface of the membrane of the bacterium, in a conformation essentially identical to that of the corresponding epitope of the natural protein G.
- the presentation of the recombinant protein on the membrane surface of the bacteria depends on its chemical nature and its peptide sequence.
- the amino acid cysteine at positions 173 and / or 186 has been replaced by an amino acid which does not form of disulfide bridge, in particular serine.
- Such a mutation promotes the formation of the disulfide bridge between the cysteine residues remaining in positions 176 and 182, which is critical for the immunogenicity of the sequence; it prevents the formation of disordered disulfide bridges (A: Figures 1 and 2).
- Peptides useful for the implementation of such a process are those comprising in particular one of the sequences ID No. 3 or ID No. 4.
- the amino acids phenylalanine corresponding to positions 163, 165, 168 and / or 170 of the sequence of protein G are replaced by an acid polar amine, in particular serine (B: Figures 1 and 2).
- polypeptide can in particular have the sequence ID No. 5.
- the suppression of the hydrophobic region located near the critical loop between the two amino acids cysteine in positions 176 and 182, allows the recombinant protein to better cross the bacterial membrane and to correctly expose the immunodominant part on the membrane surface.
- sequence of the heterologous peptide expressed in the bacterium can include the sequence ID No. 6.
- sequences having at least 80% and preferably at least 90% homology with the sequences mentioned above are suitable for the implementation of the invention. They include in particular the sequences resulting from point mutations in the nucleotide sequence and comprising the replacement of at least one amino acid by an equivalent amino acid; equivalent amino acid is understood to mean a molecule having a similar chemical structure and a similar molecular weight.
- the invention also comprises a bacterium expressing a peptide or a protein, capable of being obtained by the method described in the present application. Said bacteria can be alive or killed.
- the DNA sequence introduced into the bacterium leads to the expression of the heterologous polypeptide in the form of a fusion protein.
- a sequence coding for another peptide is then incorporated into the construction, which will act as a "spacer” or spacer and allow optimal presentation of the heterologous polypeptide on the surface of the bacteria.
- a protein which can advantageously be used as a "spacer” is a protein binding to human serum albumin, in particular the G protein of streptococcus (designated in the rest of the text by BB), or one of its fragments.
- the fusion of the heterologous polypeptide with the spacer is done by the C-terminus of said polypeptide, to promote the accessibility of the antigen to the immune system.
- Polypeptides or bacteria having one or more of the above characteristics are useful as medicaments.
- the invention comprises pharmaceutical compositions, characterized in that they comprise a polypeptide or a bacterium according to the invention in admixture with pharmaceutically acceptable adjuvants.
- the live vector oral vaccine must include the modified protein which has the optimal conformation to induce the best protection against RSV. This is why the present invention also relates to an application of such a pharmaceutical composition to the preparation of an oral vaccine intended to prevent infections caused by the respiratory syncytial virus.
- the subject of the invention is the application of such a composition to the preparation of a parenteral, enteral or mucosal vaccine intended to prevent infections caused by the respiratory syncitial virus.
- the subject of the invention is the nucleotide sequences coding for a polypeptide carried by a peptide sequence comprised between the amino acid residues 130 and 230 of the protein G of the respiratory virus or for a polypeptide having at least 80% homology with said sequence peptide, and further comprising the means allowing expression of the polypeptide on the surface of the membrane of a non-pathogenic bacterium of the genus Staphylococcus.
- Figure 1 Construction of the shuttle vector pS-'mpl 8BBXM.
- Figure 2 Construction of the shuttle vector pSPPmpl ⁇ BBXM.
- Figure 3 Extraction and analysis of surface recombinant S. xylosus membrane proteins.
- Figure 4 Analysis by flow cytometry of the recombinant proteins on the surface of S. xylosus.
- Figure 5 Analysis by flow cytometry of the recombinant proteins on the surface of S. carnosus.
- Figure 6a Antibody response induced by S. carnosus and the BB protein in BALB / c mice.
- Figure 6b Antibody response induced by S. carnosus and the BB protein in C57B1 / 6 mice.
- hybridization / ligation buffer 50mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 1 mM ATPP, 1 mM 1, 4-dithiothreitol [DTT ], 5% Polyethylene glycol [PEG] 8000.
- the hybridization mixture is incubated at 70 ° C. for 5 min and allowed to return to 37 ° C. before adding 3 units of T4 DNA ligase (BRL) followed by 15 min of incubation at 37 ° C.
- the reaction mixture is rinsed before adding 0.2 pmol of the following oligo.
- the hybridization / ligation procedure is repeated as many times as a new 5 ′ phosphorylated complementary oligo is added.
- the DNA duplex fixed on magnetic beads can be separated from the support by cutting with enzymes of suitable restrictions.
- oligonucleotide linker (5'-AGCTTGGCTG TTCCGCCATGGCTCGAG- 3 ', with the complementary strand) is inserted into the HindIII site of the plasmid pSZZmpl ⁇ XM (Hansson et al, 1992, J. Bacteriol., 174: 4239-4245), thus creating two additional restriction sites Ncol and Xhol downstream of the HindIII site of the resulting vector pSZZmpl 8 (XhoI) XM.
- a gene fragment encoding 198 amino acids, called BB, from the binding region of the serum albumin of the streptococcal protein G (Nygren et al, 1988. J. Mol.
- the fragment is digested with HindIII and Xhol and cloned downstream of the multiple cloning site mp l ⁇ of the vector pSZZmpl 8 (XhoI) XM; the resulting vector pSZZmpl 8 (XhoI) BBXM is digested with NotI and HindIII.
- the fragment containing ZZ is replaced by another fragment digested with the same vector rectification enzymes pE'mpl ⁇ (Sophia Hober, unpublished).
- the resulting vector is named pSE'mpl ⁇ BBXM ( Figure 1).
- hyieus is isolated by a SalI-HindIII digestion and ligated into the plasmid. pRIT28 previously digested with the same enzymes. The resulting plasmid designated pSLip contains the origin of both E. coli and S. aureus.
- a gene fragment coding for the C-terminal part is generated by PCR with the primers (1: S'-CCGAATTCTCGAGGCTETTAAAGAAAATAC-S 'and 2: 5'- CCAAGCTTGGATCCTGCGCAGATCTTGGTGTTGGT _TTTTG-3 ') on the plasmid template pLipPS17 thus creating two restriction sites EcoRI and Xhol downstream and Fspl, BamHI, HindIII upstream.
- the gene fragment coding for the C-terminal part of the propeptide is digested with EcoRI / HindIII and cloned into the vector pRIT28 to verify the sequence then digested XhoI / BamIII and transferred to the restriction sites SalI / BamHI of the vector pSLip.
- the resulting plasmid pSPP is digested with HindIII, complemented by the Klenow polymerase enzyme and religated to give the plasmid pSPPHindIII.
- the shuttle vector pSPPmpl ⁇ BBXM (FIG. 2) is constructed by NotI digestion of pSPPHindIII, followed by filling, digested with BamHI and ligated with a BamIII / EcoRV fragment isolated from pSE'mpl ⁇ BBXM.
- the proteins are separated on two identical SDS-PAGE gels (12%) with the precolored standard molecular size markers (Gibco BRL). A gel is colored with Coomassie Blue. The second is transferred to Problot TM membrane (Applied Biosystem) for the immunoblot with the specific anti-G l antibody (obtained from rabbit serum immunized with the peptide G l (aal 74-187) according to current protocols d 'immunization). See Figure 3.
- the cultures of the recombinant S. xylosus bacteria are made as described above.
- To build a stock solution we resuspends the bacteria in a 0.1% PBS solution of Sodium Azide (w / v) at the final concentration estimated by optical density (600nm) equal to unity.
- 30 ⁇ l of stock solution are aliquoted in each conical well of a microtiter plate, centrifuged at 550 g for 10 minutes at 4 ° C.
- the bacterial pellet is resuspended in a volume of 150 ⁇ l of PBS solution containing rabbit serum polyclonal anti-G2 (titrated 1: 1,280,000) diluted to 1 / 200th, incubation for 30 minutes.
- the bacterial cells are rinsed twice with PBS and incubated in 150 ⁇ l of a PBS solution containing anti-rabbit FITC (Sigma) diluted to 1/100 for a period of 30 minutes. After rinsing the cells twice with PBS buffer, it is resuspended in a Falcon tube containing 1 ml of PBS-Paraformaldehyde buffer 1% (w / v). The prepared samples are analyzed on the FACScanTM device (Becton Dickinson). The fluorescence distribution of each cell suspension is analyzed by the LYSIS HTM software and is represented by fluorescence histograms. See Figure 4.
- Example 5 The cultures of the recombinant S. carnosus bacteria transformed by the shuttle vector pSPPG2BBXM, or pSPPG2subBBXM, or pSPPG2delBBXM are made as described in Example 2.
- the analysis by FACscan is identical to that used in Example 3, with the only difference that the bacterial pellet is resuspended in a volume of 150 ⁇ l of PBS solution containing anti-G2 monoclonal mouse serum (860 ⁇ g / ⁇ l) diluted 1/400, incubation for 60 minutes.
- the fluorescence distribution of each cell suspension is analyzed by the LYSIS II T M software and is represented by fluorescence histograms. See figure 5.
- Example 5 Example 5
- mice received 8 oral administrations (gastric intubation), once a week, of 10 10 S. carnosus, transformed by the vector pSPPBBXM, per mouse under 0.5 ml .
- the dosage of anti-BB IgG serum antibodies is determined by ELISA test.
- the secondary antibody is revealed by depositing 100 ⁇ l of Microvvell TMB substrate in all the wells, then the enzymatic reaction is blocked by adding 100 ⁇ l of 1 M H 2 S04 to all the wells of the microplate.
- the results are read at 450 nm with the DYNATECH MR5000 or LABSYSTEMS iEMS plate reader).
- the dosage of anti-staphylococcal IgG serum antibodies is determined by ELISA as described above.
- the antigens adsorbed are whole Staphylococci at a concentration of 108 bacteria per well.
- the results are read at 450 nm with the DYNATECH MRS 000 or LABSYSTEMS iEMS plate reader).
- BALB / c mice receive 3 subcutaneous BB injections in the presence of Fréund adjuvants.
- the blood is taken individually on D38 and the anti-BB titers are determined by ELISA test as described in Example 5.
- FIG. 7 shows that 3 subcutaneous injections of 10 ng of BB do not induce an antibody response.
- 3 injections of 10 9 S. xylosus expressing BB on its surface induce an anti-BB titer of 1/10000.
- S. xylosus potentiates the immunogenicity of the antigen it expresses.
- Comassie blue staining SDS-Page gel of the fusion proteins extracted from the bacterial membrane and purified by affinity on the Albumin column of the different constructions:
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Application Number | Priority Date | Filing Date | Title |
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FR9413309 | 1994-11-07 | ||
FR9413309A FR2726577B1 (fr) | 1994-11-07 | 1994-11-07 | Procede d'obtention d'un peptide derive du virus respiratoire syncitial, polypeptide et bacterie l'exprimant, et leurs applications a titre de medicament |
PCT/FR1995/001465 WO1996014418A1 (fr) | 1994-11-07 | 1995-11-07 | Proteine g du virus respiratoire syncytial exprimee sur membrane bacterienne |
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EP0789768A1 true EP0789768A1 (de) | 1997-08-20 |
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EP95939337A Withdrawn EP0789768A1 (de) | 1994-11-07 | 1995-11-07 | Protein g von respiratory syncitial virus, exprimiert auf bakterie-membran |
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US (1) | US6130091A (de) |
EP (1) | EP0789768A1 (de) |
JP (1) | JPH10509865A (de) |
AU (1) | AU704703B2 (de) |
CA (1) | CA2204618A1 (de) |
FR (1) | FR2726577B1 (de) |
NZ (1) | NZ296563A (de) |
WO (1) | WO1996014418A1 (de) |
ZA (1) | ZA959418B (de) |
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FR2766192B1 (fr) * | 1997-07-17 | 2001-07-13 | Pf Medicament | Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie |
US6699478B1 (en) | 1997-09-19 | 2004-03-02 | Wyeth Holdings Corporation | Enhanced immune response to attachment (G) protein of Respiratory Syncytial Virus |
ES2236946T3 (es) * | 1997-09-19 | 2005-07-16 | Wyeth Holdings Corporation | Peptidos derivados de la proteina de union (g) del virus respiratorio sincitial. |
FR2798857B1 (fr) * | 1999-09-23 | 2003-06-06 | Pf Medicament | 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 |
WO2008129428A2 (en) | 2007-04-19 | 2008-10-30 | Molecular Detection Inc. | Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria |
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US5149650A (en) * | 1986-01-14 | 1992-09-22 | University Of North Carolina At Chapel Hill | Vaccines for human respiratory virus |
JPH01501357A (ja) * | 1986-01-14 | 1989-05-18 | ユニバ−シテイ・オブ・ノ−ス・カロライナ | ヒト呼吸器系ウイルス用ワクチン |
US5223254A (en) * | 1987-09-29 | 1993-06-29 | Praxis Biologics, Inc. | Respiratory syncytial virus: vaccines |
SE9101433D0 (sv) * | 1991-05-13 | 1991-05-13 | Marianne Hansson | Recombinant dna sequence and its use |
-
1994
- 1994-11-07 FR FR9413309A patent/FR2726577B1/fr not_active Expired - Fee Related
-
1995
- 1995-11-07 CA CA002204618A patent/CA2204618A1/fr not_active Abandoned
- 1995-11-07 ZA ZA959418A patent/ZA959418B/xx unknown
- 1995-11-07 US US08/836,504 patent/US6130091A/en not_active Expired - Fee Related
- 1995-11-07 AU AU41201/96A patent/AU704703B2/en not_active Ceased
- 1995-11-07 WO PCT/FR1995/001465 patent/WO1996014418A1/fr not_active Application Discontinuation
- 1995-11-07 JP JP8515109A patent/JPH10509865A/ja active Pending
- 1995-11-07 EP EP95939337A patent/EP0789768A1/de not_active Withdrawn
- 1995-11-07 NZ NZ296563A patent/NZ296563A/en unknown
Non-Patent Citations (1)
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See references of WO9614418A1 * |
Also Published As
Publication number | Publication date |
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AU4120196A (en) | 1996-05-31 |
WO1996014418A1 (fr) | 1996-05-17 |
AU704703B2 (en) | 1999-04-29 |
FR2726577A1 (fr) | 1996-05-10 |
JPH10509865A (ja) | 1998-09-29 |
CA2204618A1 (fr) | 1996-05-17 |
ZA959418B (en) | 1996-06-06 |
NZ296563A (en) | 1999-04-29 |
US6130091A (en) | 2000-10-10 |
FR2726577B1 (fr) | 1997-01-31 |
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