EP1776379A1 - Immunogenic complexes, preparation method thereof and use of same in pharmaceutical compositions - Google Patents
Immunogenic complexes, preparation method thereof and use of same in pharmaceutical compositionsInfo
- Publication number
- EP1776379A1 EP1776379A1 EP05793088A EP05793088A EP1776379A1 EP 1776379 A1 EP1776379 A1 EP 1776379A1 EP 05793088 A EP05793088 A EP 05793088A EP 05793088 A EP05793088 A EP 05793088A EP 1776379 A1 EP1776379 A1 EP 1776379A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence
- protein
- immunogen
- peptide
- rsv
- 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- Withdrawn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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
-
- 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
- A61K39/385—Haptens or antigens, bound to carriers
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—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
Definitions
- the present invention relates to a method for improving the immunogenicity of an immunogen, antigen or hapten, by coupling with a small support peptide. More particularly, the present invention relates to a process for the preparation of an immunogenic complex as well as the complexes capable of being obtained by such a process, and the use of such complexes as a medicament for increasing the immunogenicity of an immunogen .
- the invention notably comprises a support peptide coupled with a peptide derived from protein G of the Respiratory Syncytial Virus (RSV) and its use as a vaccine for the treatment of respiratory infections linked to RSV.
- RSV Respiratory Syncytial Virus
- the immune system is a network of humoral and cellular components which interact to allow the host to differentiate the self from the non-self in order to eliminate it, as well as the agents considered pathogenic. To do this, the immune system has developed two mechanisms that act in concert: innate immunity and acquired immunity.
- innate immunity are grouped physical barriers (skin, mucosa, ...), cells (monocytes / macrophages, polynuclear, NK cells, ...) and soluble factors (complement, cytokines, proteins of the acute phase, ...) involved or produced in response to an attack.
- the responses of innate immunity are rapid but are neither specific nor memorized.
- the cellular mediators of acquired immunity are the T and B lymphocytes. Their interaction allows in particular the production of immunoglobulins by the latter. Unlike the responses of innate immunity, those of acquired immunity are specific, adaptable and memorizable. Indeed, the penetration of an antigen into a new organism establishes an immune response called primary response, during which will multiply long-lived lymphocytes (T and B), called memory lymphocytes. Thanks to these cells, during a second penetration of the same antigen, the immune reaction, called secondary, will be faster and more intense. For a primary response to take place, the antigen must first be picked up and primed by the antigen presenting cells, to be presented to T cells.
- T and B long-lived lymphocytes
- vaccines The purpose of vaccines is to protect the host by preventing or limiting the invasion of pathogens.
- all vaccines on the market fulfill this role by stimulating the production of antibodies.
- carrier protein a protein having T epitopes capable of interacting with T lymphocytes.
- the best known vaccine carrier proteins are diphtheria and tetanus toxoids. Among these carrier proteins, mention may also be made of the protein known as
- BB fragment of the Streptococcus protein G, capable of binding albumin, fragment corresponding to residues 24 to 242 of the sequence SEQ ID No. 1.
- This protein makes it possible to trigger an earlier and more intense primary antibody response vis -in relation to the vaccine antigen associated with it (Libon et al., Vaccine, 17 (5): 406-41,1999). As such, reference may also be made to the international patent application published under the number WO 96/14416.
- the present invention aims to provide an alternative to carrier proteins which would, as will become apparent from the description below, overcome all of the drawbacks associated with the use of such a carrier protein. More particularly, the present invention makes it possible to limit the side effects linked to the presence of a relatively large carrier protein while allowing high production yields to be obtained.
- the inventors have highlighted an alternative to the use of carrier proteins. More particularly, the inventors have characterized a method for improving the immunogenicity of an immunogen based on the identification of a peptide, hereinafter called carrier peptide, very small in size and therefore non-immunogenic, facilitating their synthesis and / or synthesis of complexes immunogen-support peptide in which they participate.
- the present invention relates to a process for the preparation of an immunogenic complex in which an immunogen, antigen or hapten, is combined with a support peptide to form said immunogenic complex, characterized in that said support peptide consists of a peptide of less than 10 amino acids comprising at least the peptide fragment of 3 amino acid residues of sequence SEQ ID No. 2 (Met-Glu-Phe).
- immunogen any substance capable of inducing an immune response.
- the immunogen is preferably a protein, a glycoprotein a lipopeptide, or any immunogenic compound comprising in its structure a peptide of at least 5 amino acids, preferably at least 10, 15, 20, 25, 30 or 50 amino acids, compound capable of causing an immune response, in particular capable of inducing the production of specific antibodies directed against this peptide after its administration in a mammal.
- the terms polypeptides, polypeptide sequences, peptides and proteins are interchangeable.
- the expression peptide support is to be differentiated from the expression carrier protein.
- a carrier protein is characterized by a large size (218 amino acids for the BB protein) and especially the presence of T epitopes capable of binding to T receptors for the antigen present on the surface of T lymphocytes.
- the peptide support object of the present invention differs from a carrier protein because of its very small size (less than 10 amino acids) and also because it does not have T epitopes.
- the object method of the present invention makes it possible to obtain immunological complexes making it possible to improve the immunogenicity of an immunogen whose production is easier or with higher production yields.
- the complex comprising the support peptide which is the subject of the invention being much smaller in size than complexes comprising carrier proteins of the prior art, it is easier to produce by peptide / chemical synthesis or any other known technique.
- the immunogenic complexes according to the invention which are the subject of the invention make it possible to eliminate, at the very least to limit, the undesirable effects linked to the very nature of the carrier protein. It is recognized by those skilled in the art that a relatively large carrier protein, such as BB, is likely to be the basis of unwanted immune responses.
- the invention provides an advantageous alternative to the use of carrier proteins. Indeed, due to its reduced size, the support peptide has no, at least very little, chance of being the cause of side or undesirable effects.
- the support peptide of less than 10 amino acids comprises at least the peptide coded by SEQ ID No. 2 and consists of at most 8 amino acids, preferably at most 5 amino acids, and even better 4 amino acids.
- the support peptide of less than 10 amino acids which is the subject of the present invention consists of the peptide of sequence SEQ ID No. 2.
- association between said support peptide and the immunogen can be carried out by any coupling technique known to a person skilled in the art making it possible to preserve the integrity as well as the immunogenic properties of the immunogen. More particularly, the process which is the subject of the invention is characterized in that said association consists of covalent coupling.
- covalent coupling it is necessary to understand chemical coupling or protein fusion by the so-called recombinant DNA technique (fusion protein obtained after translation of a nucleic acid coding for the fusion protein (immunogenic complex) by a host cell (eukaryote or prokaryotic) transformed with said nucleic acid.
- Said support peptide can be coupled to the N-terminal or C-terminal end said immunogen when said iminunogen is a peptide.
- Preferably said support peptide is coupled to the N-terminal end of said immunogen.
- Immunogenicity can be produced by recombinant DNA techniques, in particular by insertion or fusion into the molecule of DNA coding for the support, DNA coding for the immunogen.
- the covalent coupling between the support peptide and the immunogen is carried out chemically, according to techniques known to those skilled in the art.
- the subject of the invention is also a method according to the invention in which said immunogenic complex is obtained by genetic recombination (recombinant protein) using a nucleic acid resulting from the fusion of (or insertion into) the DNA molecule coding for the support peptide with DNA coding for
- the method according to the invention comprises, in one of its modes of implementation, a step of production of the complex, by genetic engineering, in a host cell.
- the host cell can be of the prokaryotic type and be chosen in particular from the group comprising: E. coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus; it can also be a yeast.
- the host cell is a eukaryotic cell, such as a mammalian cell or an insect cell (type Sf9).
- the fusion nucleic acid encoding the immunogenic complex can in particular be introduced into the host cell via a viral vector.
- the immunogen used preferably comes from bacteria, parasites, viruses or antigens associated with tumors, such as antigens associated with melanomas or hCG beta derivatives.
- the method according to the invention is particularly suitable for a surface polypeptide of a pathogenic agent.
- a pathogenic agent When the latter is expressed in the form of a fusion protein, by recombinant DNA techniques, the fusion protein is advantageously expressed, anchored and exposed on the surface of the membrane of the host cells. Nucleic acid molecules are used which are capable of directing the synthesis of the antigen in the host cell.
- promoter sequences include promoter sequences, functionally linked secretion signal and sequence coding for a membrane anchoring region, which will be adapted by those skilled in the art.
- the immunogen can in particular be derived from a surface glycoprotein of human RSV type A or B, or from bovine RSV, in particular chosen from proteins F and G.
- the immunogen consists of a polypeptide encoded by a sequence between residues 130-230 of the peptide sequence of the G protein of RSV or by any sequence having at least 80% identity with said peptide sequence, preferably 85%, 90%, 95% or 98% identity with the sequence between residues 130-230 of the peptide sequence of said protein G, or one of its fragments of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies directed against this fragment after its administration in a mammal.
- percentage of identity or “percentage of homology” (the two expressions being used interchangeably in the present description) between two nucleic acid or amino acid sequences within the meaning of the present invention, is intended to denote a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment (optimal 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 aligned them optimally, said comparison being able to be carried out by segment or by “comparison window”.
- the optimal alignment of the sequences for comparison can be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).
- the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid or amino acid sequence to be compared may include additions or deletions compared 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.
- BLAST 2 sequences (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters "open gap penaltie”: 5, and “extension gap penaltie”: 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
- amino acid sequence having at least 80%, preferably 85%,
- the method according to the invention is characterized in that the immunogen is the polypeptide of sequence SEQ ID No. 3, or of sequence having at least 80% identity with the sequence SEQ ID No. 3, preferably 85%, 90%, 95% or 98% identity with the sequence between residues 130-230 of the peptide sequence of said G protein, or one of the fragments of the sequence SEQ ID N 0 3 of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies directed against this fragment after its administration in a mammal.
- immunogens adapted to the implementation of the method according to the invention comprise a derivative of the surface protein of the hepatitis A virus, B and C, a surface protein of the measles virus, a surface protein parainffuenza 3 virus, in particular a surface glycoprotein such as hemaglutinin, neuraminidase HN and the fusion protein F.
- the present invention relates to an immunogenic complex obtained by the implementation of the method according to the invention.
- the present invention also relates to an immunogenic complex, comprising an immunogen, an antigen or a hapten, characterized in that said immunogen is associated with a support peptide of less than 10 amino acids comprising at least the peptide fragment of 3 amino acid residues of sequence SEQ ID No. 2.
- said support peptide comprising at least the peptide coded by SEQ ID No. 2 consists of at most 8 amino acids, preferably at most 5 amino acids, and even better of 4 amino acids.
- said peptide supporting the immunogenic complex according to the invention consists of the peptide coded by SEQ ID No. 2.
- said support peptide of the immunogenic complex according to the invention is characterized in that said association consists of a covalent coupling between said support peptide and said immunogen.
- said immunogenic complex according to the invention is characterized in that said support peptide is coupled to the N- or C-terminal end of said immunogen when said immunogen is a peptide, preferably N-terminal.
- said immunogenic complex according to the invention is characterized in that Pirnrnunorelates is an antigen derived from bacteria, parasites and / or viruses.
- said immunogenic complex according to the invention is characterized in that the immunogen is a surface protein or glycoprotein, in particular F or G, of the respiratory syncytial virus (RSV), or of sequence having at least 80% identity with the sequence of said protein F or G, preferably 85%,
- RSV respiratory syncytial virus
- said immunogenic complex according to the invention is characterized in that the immunogen is protein G of human RSV type A or B, protein G of bovine RSV.
- said immunogenic complex according to the invention is characterized in that the immunogen is the polypeptide of sequence between residues 130-230 of protein G of RSV, ends included, or of sequence having at least 80% identity with said sequence 130-230, or one of the fragments of at least 10 amino acids of said sequence 130-230 of protein G of RSV.
- the immunogen of said immunogenic complex according to the invention is the polypeptide of sequence SEQ ID No. 3.
- the complex according to the invention is the MEFG2Na complex of sequence SEQ ID No. 4, or an analogous immunogenic complex whose sequence has in position 1 to 3 the MEF sequence of sequence SEQ ID N ° 2 followed:
- the subject of the present invention is a nucleic acid, preferably isolated and / or purified, coding for the immunogenic complexes according to the invention, in particular for the immunogenic complex MEFG2Na of sequence SEQ ID NO: 1
- nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs.
- the subject of the present invention is the immunogenic complexes according to the invention or the nucleic acids coding for the immunogenic complexes according to the invention as a medicament, in particular the complex MEFG2Na immunogen of sequence SEQ ID No. 4 or nucleic acid such as DNA or RNA, coding for this MEFG2Na complex.
- compositions comprising the immunogenic complexes according to the invention or as defined above, or a nucleic acid, RNA or DNA, coding for such immunogenic complexes, associated with physiologically acceptable excipients also form part of the invention. They are particularly suitable for the preparation of a vaccine.
- Immunization may be obtained by the administration of said polynucleotide encoding the immunogenic complexes as defined above, alone or through a viral vector comprising such a polynucleotide. It is also possible to use a host cell, in particular an inactivated bacterium, transformed with such a polynucleotide according to the invention.
- the present invention also relates to the use of an immunogenic complex according to the invention, in which complex said immunogen is a protein or a peptide derived from the G or F protein of RSV as defined above, in particular the MEFG2Na complex or the one of its analogs according to the invention, or a nucleic acid according to the invention encoding said immunogenic complex, for the preparation of a pharmaceutical composition intended for the prevention or treatment of respiratory infections linked to RSV.
- FIG. 1 shows the level of anti-RSV-A IgG in mice immunized with BBG2Na or MEFG2Na;
- FIG. 2 also shows, according to another presentation, the level of anti-RSV-A IgG in mice immunized with BBG2Na or MEFG2Na after 2 immunizations;
- FIG. 3 shows the level of anti-G2Na IgG in mice immunized with BBG2Na or MEFG2Na;
- FIG. 4 also shows, according to another presentation, the level of anti-G2Na IgG in mice immunized with BBG2Na or MEFG2Na.
- Example 1 Comparison of the In Vivo Activities Induced by the Use of the BB Carrier Protein or of the MEF Support Peptide
- mice 8-week-old female IOPS BALB / c mice are infected nasally with RSV-A Long strain (10 5 pfu) at D-20.
- RSV-A Long strain 10 5 pfu
- the mice receive a single intramuscular injection of 20 ⁇ g of BBG2Na adsorbed on Adju-Phos (i.e. 6 ⁇ g equivalent G2Na) or 6 ⁇ g of MEFG2Na adsorbed on Adju-Phos .
- the level of anti-RSV-A IgG (purified viral antigen) and anti-MEFG2Na is monitored by ELISA.
- Figures 1 and 2 show that there is no significant difference between the level of anti-RSV-A IgG triggered by 6 ⁇ g of MEFG2Na or 20 ⁇ g of BBG2Na, and this at no point in the kinetics. The same is true for the anti-G2Na IgG level ( Figures 3 and 4).
- Example 2 Preparation of BBG2Na and MEFG2Na complexes
- the BBG2Na protein is produced using Escherichia coli RV308 as host cell and a plasmid where the transcription of the gene of interest is under the control of the trytophane promoter.
- the fermentation stage is a batch type process with a semi-defined synthetic culture medium and glycerol as a source of carbon and energy. Two culture steps are necessary to prepare the inoculum which is used to seed the production fermenter.
- the microorganisms are cultured up to an optical density at 620 nm of 50, then expression is induced by the addition of a tryptophan analog (IAA).
- IAA tryptophan analog
- the culture is continued until the partial pressure of O 2 in the fermenter suddenly rises, which signals the exhaustion of the carbon source.
- the average cell density is 40 g of dry cells / liter with an expression rate of
- the extraction of BBG2Na requires solubilization of the pellet of thawed microorganisms with a buffer containing guanidine, HCl and 1,4-dithiothreitol (DTT) to reduce the disulfide bridges.
- the renaturation of the protein and the oxidation of the disulfide bridges are obtained by diluting the denatured suspension and stirring at room temperature overnight in an open reactor.
- the suspension containing the renatured protein is clarified by centrifugation and then filtered.
- PEG 6000 is added to the filtrate and the resulting precipitate is recovered by centrifugation.
- the precipitate containing BBG2Na is solubilized again in a buffer containing urea.
- the extract obtained is filtered through a 0.22 ⁇ m support and stored at -15 / -25 ° C.
- the purification of BBG2Na from the thawed extract comprises five stages which are: (1) cation exchange chromatography on a column of SP-Sepharose fast flow, (2) hydrophobic interaction chromatography on a column of methyl Macroprep, (3) a gel filtration on a column of Superdex S200, (4) an anion exchange chromatography on a column of DEAE-Sepharose and finally (5) a desalting step on a column of Sephadex G25.
- the purified protein solution is sterile filtered and distributed in sterile and pyrogen-free bags.
- MEFG2Na protein is produced using Escherichia coli ICONE 200 as host cell and a plasmid where the transcription of the gene of interest is under the control of the trytophan promoter.
- E. coli ICONE 200 is a mutant of E. coli RV308 which was developed to improve expression control during the growth phase.
- the fermentation step is a fed-batch type process with a chemically defined culture medium and glycerol as a source of carbon and energy. Two culture steps are necessary to prepare the inoculum which is used to seed the production fermenter.
- the microorganisms are grown up to an optical density at 620 nm of 110, then expression is induced by the addition of a tryptophan analog (IAA).
- IAA tryptophan analog
- the culture is continued until the partial pressure of O 2 in the fermenter suddenly rises, which signals the exhaustion of the carbon source.
- the average cell density is 56 g of dry cells / liter with an expression rate of 5.4%, ie a productivity of 3 g of MEFG2Na / liter of culture.
- the culture is cooled to + 4 ° C., the microorganisms are recovered by centrifugation and frozen at -15 / -25 ° C.
- MEFG2Na The extraction of MEFG2Na requires solubilization of the pellet of thawed micro ⁇ organisms with a buffer containing guanidine, HCl. Suspension containing the denatured protein is clarified by centrifugation and then filtered. Since guanidine is incompatible with the subsequent purification step, a dialysis concentration step, on a polyethersulfone ultrafiltration support with a cutoff threshold of 10 kDa, is put in place to effect the change of buffer. The extract obtained is filtered on a 0.22 ⁇ m support and purified in stride.
- the purification of MEFG2Na comprises three stages which are: (1) cation exchange chromatography on a column of Fractogel EMD SE Hicap, (2) gel filtration on a column of Superdex 75 Prep Grade, (3) chromatography of exchange of anions on a column of DEAE Sepharose Fast Flow.
- the bulk of purified protein is sterile filtered and distributed in sterile and pyrogen-free bags.
- the level of expression of the MEFG2Na complex is approximately twice as high as the level of expression of the BBG2Na complex.
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Abstract
The invention relates to a method of improving the immunogenicity of an immunogen, antigen or hapten, by means of coupling with a small support peptide. More specifically, the invention relates to a method of preparing an immunogenic complex, as well as the complexes that can be obtained using one such method, and to the use of said complexes as a medicament in order to increase the immunogenicity of an immunogen. The invention comprises, for example, a support peptide which is coupled with a peptide from protein G of the respiratory syncytial virus (RSV) and to the use thereof as a vaccine for the treatment of respiratory infections linked to RSV.
Description
COMPLEXES IMMUNOGENES, LEUR PROCEDE DE PREPARATION ET LEUR UTILISATION DANS DES COMPOSITIONS PHARMACEUTIQUESIMMUNOGENIC COMPLEXES, THEIR PREPARATION PROCESS AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS
La présente invention concerne un procédé permettant d'améliorer l'immunogénicité d'un immunogène, antigène ou haptène, par couplage avec un peptide support de petite taille. Plus particulièrement, la présente invention concerne un procédé de préparation d'un complexe immunogène ainsi que les complexes susceptibles d'être obtenus par un tel procédé, et l'utilisation de tels complexes à titre de médicament pour augmenter l'immunogénicité d'un immunogène. L'invention comprend notamment un peptide support couplé avec un peptide issu de la protéine G du Virus Respiratoire Syncytial (VRS) et son utilisation à titre vaccinal pour le traitement des infections respiratoires liées au VRS.The present invention relates to a method for improving the immunogenicity of an immunogen, antigen or hapten, by coupling with a small support peptide. More particularly, the present invention relates to a process for the preparation of an immunogenic complex as well as the complexes capable of being obtained by such a process, and the use of such complexes as a medicament for increasing the immunogenicity of an immunogen . The invention notably comprises a support peptide coupled with a peptide derived from protein G of the Respiratory Syncytial Virus (RSV) and its use as a vaccine for the treatment of respiratory infections linked to RSV.
Le système immunitaire est un réseau de composants de nature humorale et cellulaire qui interagissent pour permettre à l'hôte de différencier le soi du non-soi afin de l'éliminer, ainsi que les agents jugés pathogènes. Pour ce faire, le système immunitaire a développé deux mécanismes qui agissent de concert : immunité innée et immunité acquise.The immune system is a network of humoral and cellular components which interact to allow the host to differentiate the self from the non-self in order to eliminate it, as well as the agents considered pathogenic. To do this, the immune system has developed two mechanisms that act in concert: innate immunity and acquired immunity.
Sous le terme d'immunité innée sont regroupés les barrières physiques (peau, muqueuse, ...), les cellules (monocytes/macrophages, polynucléaires, cellules NK, ...) et les facteurs solubles (complément, cytokines, protéines de la phase aiguë, ...) mis en jeu ou produits en réponse à une agression. Les réponses de l'immunité innée sont rapides mais ne sont ni spécifiques ni mémorisées.Under the term innate immunity are grouped physical barriers (skin, mucosa, ...), cells (monocytes / macrophages, polynuclear, NK cells, ...) and soluble factors (complement, cytokines, proteins of the acute phase, ...) involved or produced in response to an attack. The responses of innate immunity are rapid but are neither specific nor memorized.
Les médiateurs cellulaires de l'immunité acquise sont les lymphocytes T et B. Leur interaction permet notamment la production d'immunoglobulines par ces derniers. A l'inverse des réponses de l'immunité innée, celles de l'immunité acquise sont spécifiques, adaptables et mémorisables. En effet, la pénétration d'un antigène dans un organisme neuf instaure une réponse immunitaire dite réponse primaire, au cours de laquelle se multiplieront des lymphocytes (T et B) à vie longue, appelés lymphocytes à mémoire. Grâce à ces cellules, lors d'une deuxième pénétration du même antigène, la réaction immunitaire, dite secondaire, sera plus rapide et plus intense. Pour qu'une réponse primaire ait lieu, il faut tout d'abord que l'antigène soit capté et apprêté par les
cellules présentant l'antigène, pour être présenté aux lymphocytes T.The cellular mediators of acquired immunity are the T and B lymphocytes. Their interaction allows in particular the production of immunoglobulins by the latter. Unlike the responses of innate immunity, those of acquired immunity are specific, adaptable and memorizable. Indeed, the penetration of an antigen into a new organism establishes an immune response called primary response, during which will multiply long-lived lymphocytes (T and B), called memory lymphocytes. Thanks to these cells, during a second penetration of the same antigen, the immune reaction, called secondary, will be faster and more intense. For a primary response to take place, the antigen must first be picked up and primed by the antigen presenting cells, to be presented to T cells.
Les vaccins ont pour but de protéger l'hôte en empêchant ou limitant l'invasion d'agents pathogènes. A l'heure actuelle, tous les vaccins commercialisés remplissent ce rôle en suscitant la production d'anticorps. Lorsque l'antigène vaccinant n'est pas à même de déclencher une réponse immunitaire ou que celle-ci est trop faible, son association physique à une protéine, dite protéine porteuse, possédant des épitopes T capables d'interagir avec les lymphocytes T permet d'y suppléer. Les protéines porteuses vaccinales les plus connues sont les anatoxines diphtériques et tétaniques. Parmi ces protéines porteuses, on peut également citer la protéine dénomméeThe purpose of vaccines is to protect the host by preventing or limiting the invasion of pathogens. Currently, all vaccines on the market fulfill this role by stimulating the production of antibodies. When the vaccinating antigen is not able to trigger an immune response or that it is too weak, its physical association with a protein, called carrier protein, having T epitopes capable of interacting with T lymphocytes allows 'make up for it. The best known vaccine carrier proteins are diphtheria and tetanus toxoids. Among these carrier proteins, mention may also be made of the protein known as
« BB », fragment de la protéine G des Streptocoques, capable de lier l'albumine, fragment correspondant aux résidus 24 à 242 de la séquence SEQ ID N° 1. Cette protéine permet de déclencher une réponse anticorps primaire plus précoce et plus intense vis-à-vis de l'antigène vaccinant qui lui est associé (Libon et al., Vaccine, 17(5): 406-41,1999). A ce titre, on pourra également se référer à la demande internationale de brevet publiée sous le numéro WO 96/14416."BB", fragment of the Streptococcus protein G, capable of binding albumin, fragment corresponding to residues 24 to 242 of the sequence SEQ ID No. 1. This protein makes it possible to trigger an earlier and more intense primary antibody response vis -in relation to the vaccine antigen associated with it (Libon et al., Vaccine, 17 (5): 406-41,1999). As such, reference may also be made to the international patent application published under the number WO 96/14416.
La présente invention a pour but de fournir une alternative aux protéines porteuses qui permettrait, comme il ressortira de la description ci-après, de remédier à l'ensemble des inconvénients liés à l'utilisation d'une telle protéine porteuse. Plus particulièrement, la présente invention permet de limiter les effets secondaires liés à la présence d'une protéine porteuse de relativement grande taille tout en permettant l'obtention de rendements de productions élevés.The present invention aims to provide an alternative to carrier proteins which would, as will become apparent from the description below, overcome all of the drawbacks associated with the use of such a carrier protein. More particularly, the present invention makes it possible to limit the side effects linked to the presence of a relatively large carrier protein while allowing high production yields to be obtained.
Dans un souci de clarté, les avantages de la présente invention seront mis en évidence en comparaison avec une protéine porteuse de l'état de la technique, à savoir la protéine porteuse BB.For the sake of clarity, the advantages of the present invention will be highlighted in comparison with a carrier protein of the prior art, namely the BB carrier protein.
De manière tout à fait inattendue, et contrairement aux connaissances actuelles et admises de l'homme de l'art, les inventeurs ont mis en évidence une alternative à l'utilisation de protéines porteuses. Plus particulièrement, les inventeurs ont caractérisé un procédé d'amélioration de l'immunogénicité d'un immunogène reposant sur l'identification d'un peptide, appelé ci-après peptide support, de très petite taille et par conséquent non immunogène, facilitant leur synthèse et/ou la synthèse des complexes
immunogènes-peptide support auxquels ils participent.Quite unexpectedly, and contrary to current and accepted knowledge of those skilled in the art, the inventors have highlighted an alternative to the use of carrier proteins. More particularly, the inventors have characterized a method for improving the immunogenicity of an immunogen based on the identification of a peptide, hereinafter called carrier peptide, very small in size and therefore non-immunogenic, facilitating their synthesis and / or synthesis of complexes immunogen-support peptide in which they participate.
Pour ce faire, la présente invention concerne un procédé de préparation d'un complexe immunogène dans lequel on associe un immunogène, antigène ou haptène, à un peptide support pour former ledit complexe immunogène, caractérisé en ce que ledit peptide support consiste en un peptide de moins de 10 acides-aminés comprenant au moins le fragment peptidique de 3 résidus d'acide aminé de séquence SEQ ID N° 2 (Met-Glu-Phe).To do this, the present invention relates to a process for the preparation of an immunogenic complex in which an immunogen, antigen or hapten, is combined with a support peptide to form said immunogenic complex, characterized in that said support peptide consists of a peptide of less than 10 amino acids comprising at least the peptide fragment of 3 amino acid residues of sequence SEQ ID No. 2 (Met-Glu-Phe).
Par immunogène, il faut comprendre toute substance capable d'entraîner une réponse immunitaire. A titre d'exemple non limitatif, Pimmunogène est de préférence une protéine, une glycoprotéine un lipopeptide, ou tout composé immunogène comprenant dans sa structure un peptide d'au moins 5 acides aminés, de préférence d'au moins 10, 15, 20, 25, 30 ou 50 acides aminés, composé capable d'entraîner une réponse immunitaire, notamment capable d'induire la production d'anticorps spécifiques dirigés contre ce peptide après son administration chez un mammifère. Dans la présente description, les termes polypeptides, séquences polypeptidiques, peptides et protéines sont interchangeables.By immunogen is meant any substance capable of inducing an immune response. By way of nonlimiting example, the immunogen is preferably a protein, a glycoprotein a lipopeptide, or any immunogenic compound comprising in its structure a peptide of at least 5 amino acids, preferably at least 10, 15, 20, 25, 30 or 50 amino acids, compound capable of causing an immune response, in particular capable of inducing the production of specific antibodies directed against this peptide after its administration in a mammal. In the present description, the terms polypeptides, polypeptide sequences, peptides and proteins are interchangeable.
Au regard de ce qui a été décrit plus haut, il faut bien comprendre que l'expression peptide support est à différencier de l'expression protéine porteuse. En effet, une protéine porteuse se caractérise par une taille importante (218 acides aminés pour la protéine BB) et surtout la présence d'épitopes T capables de se lier aux récepteurs T pour l'antigène présents à la surface des lymphocytes T. Le peptide support objet de la présente invention diffère d'une protéine porteuse du fait de sa taille très réduite (moins de 10 acides aminés) et également du fait qu'il ne présente pas d'épitopes T. Selon un premier aspect avantageux, le procédé objet de la présente invention permet d'obtenir des complexes immunologiques permettant d'améliorer l'immunogénicité d'un immunogène dont la production est plus aisée ou avec des rendements de production plus importants. En effet, le complexe comprenant le peptide support objet de l'invention étant de bien plus petite taille que des complexes comprenant des protéines porteuses de l'art antérieur, il est plus facile à produire par synthèse peptidique/chimique ou toute autre technique connue de l'homme de l'art.
Selon un deuxième aspect avantageux, les complexes immunogènes selon l'invention objet de l'invention permettent d'éliminer, à tout le moins de limiter, les effets indésirables liés à la nature même de la protéine porteuse. Il est admis de l'homme de l'art qu'une protéine porteuse, comme BB, de taille relativement importante a de fortes chances d'être à la base de réponses immunitaires non souhaitées. Par exemple, il a été montré pour l'anatoxine tétanique que la sensibilisation préalable de l'hôte à cette protéine porteuse pouvait empêcher le développement d'une réponse anticorps contre l'antigène associé à l'anatoxine tétanique lors de la vaccination avec un conjugué (Kaliyaperumal et al, Eur. J. Immunol., 25(12) : 3375-80, 1995). Ce phénomène est connu sous le terme de suppression épitopique.In view of what has been described above, it must be understood that the expression peptide support is to be differentiated from the expression carrier protein. Indeed, a carrier protein is characterized by a large size (218 amino acids for the BB protein) and especially the presence of T epitopes capable of binding to T receptors for the antigen present on the surface of T lymphocytes. The peptide support object of the present invention differs from a carrier protein because of its very small size (less than 10 amino acids) and also because it does not have T epitopes. According to a first advantageous aspect, the object method of the present invention makes it possible to obtain immunological complexes making it possible to improve the immunogenicity of an immunogen whose production is easier or with higher production yields. In fact, the complex comprising the support peptide which is the subject of the invention being much smaller in size than complexes comprising carrier proteins of the prior art, it is easier to produce by peptide / chemical synthesis or any other known technique. one skilled in the art. According to a second advantageous aspect, the immunogenic complexes according to the invention which are the subject of the invention make it possible to eliminate, at the very least to limit, the undesirable effects linked to the very nature of the carrier protein. It is recognized by those skilled in the art that a relatively large carrier protein, such as BB, is likely to be the basis of unwanted immune responses. For example, it has been shown for tetanus toxoid that prior sensitization of the host to this carrier protein could prevent the development of an antibody response against the antigen associated with tetanus toxoid during vaccination with a conjugate (Kaliyaperumal et al, Eur. J. Immunol., 25 (12): 3375-80, 1995). This phenomenon is known as epitopic suppression.
Par conséquent, il ressort clairement de la présente description que l'invention apporte une alternative avantageuse à l'utilisation de protéines porteuses. En effet, de par sa taille réduite, le peptide support n'a aucune, à tout le moins très peu, de chance d'être à l'origine d'effets secondaires ou indésirables. Selon une forme d'exécution préférée de la présente invention, le peptide support de moins de 10 acides aminés comprend au moins le peptide codé par la SEQ ID N° 2 et est constitué d'au plus 8 acides aminés, préférentiellement d'au plus 5 acides aminés, et encore mieux 4 acides aminés.Consequently, it is clear from the present description that the invention provides an advantageous alternative to the use of carrier proteins. Indeed, due to its reduced size, the support peptide has no, at least very little, chance of being the cause of side or undesirable effects. According to a preferred embodiment of the present invention, the support peptide of less than 10 amino acids comprises at least the peptide coded by SEQ ID No. 2 and consists of at most 8 amino acids, preferably at most 5 amino acids, and even better 4 amino acids.
Selon encore une forme d'exécution plus préférée, le peptide support de moins de 10 acides aminés objet de la présente invention consiste en le peptide de séquence SEQ ID N° 2.According to yet a more preferred embodiment, the support peptide of less than 10 amino acids which is the subject of the present invention consists of the peptide of sequence SEQ ID No. 2.
L'association entre ledit peptide support et l'immunogène peut être effectuée par toute technique de couplage connue de l'homme de l'art permettant de préserver l'intégrité ainsi que les propriétés immunogéniques de l'immunogène. Plus particulièrement, le procédé objet de l'invention est caractérisé en ce que ladite association consiste en un couplage covalent. Par couplage covalent, il faut comprendre couplage chimique ou encore fusion protéique par la technique dite de l'ADN recombinant (protéine de fusion obtenue après traduction d'un acide nucléique codant pour la protéine de fusion (complexe immunogène) par une cellule hôte (eucaryote ou procaryote) transformée avec ledit acide nucléique.The association between said support peptide and the immunogen can be carried out by any coupling technique known to a person skilled in the art making it possible to preserve the integrity as well as the immunogenic properties of the immunogen. More particularly, the process which is the subject of the invention is characterized in that said association consists of covalent coupling. By covalent coupling, it is necessary to understand chemical coupling or protein fusion by the so-called recombinant DNA technique (fusion protein obtained after translation of a nucleic acid coding for the fusion protein (immunogenic complex) by a host cell (eukaryote or prokaryotic) transformed with said nucleic acid.
Ledit peptide support peut être couplé à l'extrémité N-terminale ou C-terminale
dudit immunogène lorsque ledit iminunogène est un peptide. De préférence ledit peptide support est couplé à l'extrémité N-terminale dudit immunogène.Said support peptide can be coupled to the N-terminal or C-terminal end said immunogen when said iminunogen is a peptide. Preferably said support peptide is coupled to the N-terminal end of said immunogen.
Le complexe entre le peptide support et le composé dont on souhaite améliorerThe complex between the support peptide and the compound which we wish to improve
Pimmunogénicité peut être produit par les techniques d'ADN recombinant, notamment par insertion ou fusion dans la molécule d'ADN codant pour le support, de l'ADN codant pour l' immunogène.Immunogenicity can be produced by recombinant DNA techniques, in particular by insertion or fusion into the molecule of DNA coding for the support, DNA coding for the immunogen.
Selon un autre mode de mise en oeuvre le couplage covalent entre le peptide support et l' immunogène est réalisé par voie chimique, selon des techniques connues de l'homme du métier. L'invention a également pour objet un procédé selon l'invention dans lequel ledit complexe immunogène est obtenu par recombinaison génétique (protéine recombinante) à l'aide d'un acide nucléique résultant de la fusion de (ou de l'insertion dans) la molécule d'ADN codant pour le peptide support avec l'ADN codant pourAccording to another embodiment, the covalent coupling between the support peptide and the immunogen is carried out chemically, according to techniques known to those skilled in the art. The subject of the invention is also a method according to the invention in which said immunogenic complex is obtained by genetic recombination (recombinant protein) using a nucleic acid resulting from the fusion of (or insertion into) the DNA molecule coding for the support peptide with DNA coding for
F immunogène, le cas échéant avec un promoteur. Dans ce procédé, il pourra être mis en oeuvre un vecteur contenant un tel acide nucléique de fusion, ledit vecteur pouvant avoir notamment pour origine un vecteur d'ADN qui provient d'un plasmide, d'un bactériophage, d'un virus et/ou d'un cosmide, et l'acide nucléique de fusion codant pour ledit complexe peut être intégrée dans le génome d'une cellule hôte pour y être exprimé. Ainsi le procédé selon l'invention comprend, dans l'un de ses modes de mise en oeuvre, une étape de production du complexe, par génie génétique, dans une cellule hôte.F immunogenic, if necessary with a promoter. In this process, it is possible to use a vector containing such a fusion nucleic acid, said vector possibly originating in particular from a DNA vector which comes from a plasmid, a bacteriophage, a virus and / or of a cosmid, and the fusion nucleic acid encoding said complex can be integrated into the genome of a host cell to be expressed there. Thus the method according to the invention comprises, in one of its modes of implementation, a step of production of the complex, by genetic engineering, in a host cell.
La cellule hôte peut être de type procaryote et être notamment choisie dans le groupe comprenant : E. coli, Bacillus, Lactobacillus, Staphylococcus et Streptococcus ; il peut également s'agir d'une levure.The host cell can be of the prokaryotic type and be chosen in particular from the group comprising: E. coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus; it can also be a yeast.
Selon un autre aspect, la cellule hôte est une cellule eucaryote, telle qu'une cellule de mammifère ou une cellule d'insecte (type Sf9).According to another aspect, the host cell is a eukaryotic cell, such as a mammalian cell or an insect cell (type Sf9).
L'acide nucléique de fusion codant pour le complexe immunogène peut notamment être introduit dans la cellule hôte par l'intermédiaire d'un vecteur viral. L'immunogène utilisé provient de préférence de bactéries, de parasites, de virus ou d'antigènes associés aux tumeurs, comme les antigènes associés aux mélanomes ou
dérivés de la béta hCG.The fusion nucleic acid encoding the immunogenic complex can in particular be introduced into the host cell via a viral vector. The immunogen used preferably comes from bacteria, parasites, viruses or antigens associated with tumors, such as antigens associated with melanomas or hCG beta derivatives.
Le procédé selon l'invention est particulièrement approprié pour un polypeptide de surface d'un agent pathogène. Lorsque celui-ci est exprimé sous forme de protéine de fusion, par les techniques d'ADN recombinant, la protéine de fusion est avantageusement exprimée, ancrée et exposée à la surface de la membrane des cellules hôtes. On utilise des molécules d'acides nucléiques qui sont capables de diriger la synthèse de l'antigène dans la cellule hôte.The method according to the invention is particularly suitable for a surface polypeptide of a pathogenic agent. When the latter is expressed in the form of a fusion protein, by recombinant DNA techniques, the fusion protein is advantageously expressed, anchored and exposed on the surface of the membrane of the host cells. Nucleic acid molecules are used which are capable of directing the synthesis of the antigen in the host cell.
Elles comprennent des séquences promoteur, signal de sécrétion liée de façon fonctionnelle et séquence codant pour une région d'ancrage membranaire, qui seront adaptées par l'homme du métier.They include promoter sequences, functionally linked secretion signal and sequence coding for a membrane anchoring region, which will be adapted by those skilled in the art.
L'immunogène peut notamment dériver d'une glycoprotéine de surface du VRS humain de type A ou B, ou du VRS bovin, notamment choisie parmi les protéines F et G.The immunogen can in particular be derived from a surface glycoprotein of human RSV type A or B, or from bovine RSV, in particular chosen from proteins F and G.
Des résultats particulièrement avantageux sont obtenus avec des fragments de la protéine G du VRS humain, sous-groupes A ou B, ou bovin.Particularly advantageous results are obtained with fragments of protein G of human RSV, subgroups A or B, or bovine.
De manière préférée, l'immunogène consiste en un polypeptide codé par une séquence comprise entre les résidus 130-230 de la séquence peptidique de la protéine G du VRS ou par toute séquence présentant au moins 80 % d'identité avec ladite séquence peptidique, de préférence 85 %, 90 %, 95 % ou 98 % d'identité avec la séquence comprise entre les résidus 130-230 de la séquence peptidique de ladite protéine G, ou l'un de ses fragments d'au moins 10 acides aminés consécutifs, de préférence d'au moins 15, 20, 25, 30 ou 50 acides aminés, capable d'induire la production d'anticorps spécifiques dirigés contre ce fragment après son administration chez un mammifère.Preferably, the immunogen consists of a polypeptide encoded by a sequence between residues 130-230 of the peptide sequence of the G protein of RSV or by any sequence having at least 80% identity with said peptide sequence, preferably 85%, 90%, 95% or 98% identity with the sequence between residues 130-230 of the peptide sequence of said protein G, or one of its fragments of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies directed against this fragment after its administration in a mammal.
Par « pourcentage d'identité » ou « pourcentage d'homologie » (les deux expressions étant utilisées indifféremment dans la présente description) entre deux séquences d'acides nucléiques ou d'acides aminés au sens de la présente invention, on entend désigner un pourcentage de nucléotides ou de résidus d'acides aminés identiques entre les deux séquences à comparer, obtenu après le meilleur alignement (alignement optimal), ce pourcentage étant purement statistique et les différences entre les deux séquences étant réparties au hasard et sur toute leur longueur. Les comparaisons de séquences entre deux séquences d'acides nucléiques ou d'acides aminés sont
traditionnellement réalisées en comparant ces séquences après les avoir alignées de manière optimale, ladite comparaison pouvant être réalisée par segment ou par « fenêtre de comparaison ». L'alignement optimal des séquences pour la comparaison peut être réalisé, outre manuellement, au moyen de l'algorithme d'homologie locale de Smith et Waterman (1981) [Ad. App. Math. 2:482], au moyen de l'algorithme d'homologie locale de Neddleman et Wunsch (1970) [J. Mol. Biol. 48:443], au moyen de la méthode de recherche de similarité de Pearson et Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444], au moyen de logiciels informatiques utilisant ces algorithmes (GAP, BESTFIT, FASTA et TFASTA dans le Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, ou encore par les logiciels de comparaison BLAST N ou BLAST P).By “percentage of identity” or “percentage of homology” (the two expressions being used interchangeably in the present description) between two nucleic acid or amino acid sequences within the meaning of the present invention, is intended to denote a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment (optimal 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 aligned them optimally, said comparison being able to be carried out by segment or by “comparison window”. The optimal alignment of the sequences for comparison can be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).
Le pourcentage d'identité entre deux séquences d'acides nucléiques ou d'acides aminés est déterminé en comparant ces deux séquences alignées de manière optimale dans laquelle la séquence d'acides nucléiques ou d'acides aminés à comparer peut comprendre des additions ou des délétions par rapport à la séquence de référence pour un alignement optimal entre ces deux séquences. Le pourcentage d'identité est calculé en déterminant le nombre de positions identiques pour lesquelles le nucléotide ou le résidu d'acide aminé est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions dans la fenêtre de comparaison et en multipliant le résultat obtenu par 100 pour obtenir le pourcentage d'identité entre ces deux séquences.The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid or amino acid sequence to be compared may include additions or deletions compared 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.
Par exemple, on pourra utiliser le programme BLAST, « BLAST 2 séquences » (Tatusova et al., "Blast 2 séquences - a new tool for comparing protein and nucléotide séquences", FEMS Microbiol Lett. 174:247-250) disponible sur le site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, les paramètres utilisés étant ceux donnés par défaut (en particulier pour les paramètres « open gap penaltie » : 5, et « extension gap penaltie » : 2 ; la matrice choisie étant par exemple la matrice « BLOSUM 62 » proposée par le programme), le pourcentage d'identité entre les deux séquences à comparer étant calculé directement par le programme. Par séquence d'acides aminés présentant au moins 80 %, de préférence 85 %,For example, one could use the BLAST program, "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters "open gap penaltie": 5, and "extension gap penaltie": 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program. By amino acid sequence having at least 80%, preferably 85%,
90 %, 95 % et 98 % d'identité avec une séquence d'acides aminés de référence, on
préfère celles présentant par rapport à la séquence de référence, certaines modifications, en particulier une délétion, addition ou substitution d'au moins un acide aminé, une troncation ou un allongement. Dans le cas d'une substitution, d'un ou plusieurs acide(s) aminé(s) consécutifs) ou non consécutifs), on préfère les substitutions dans lesquelles les acides aminés substitués sont remplacés par des acides aminés « équivalents ». L'expression « acides aminés équivalents » vise ici à désigner tout acide aminé susceptible d'être substitué à l'un des acides aminés de la structure de base sans cependant modifier essentiellement les activités biologiques des anticorps correspondants. Ces acides aminés équivalents peuvent être déterminés soit en s 'appuyant sur leur homologie de structure avec les acides aminés auxquels ils se substituent, soit sur des résultats d'essais comparatifs d'activité biologique entre les différents anticorps susceptibles d'être effectués.90%, 95% and 98% identity with a reference amino acid sequence, we prefers those presenting with respect to the reference sequence, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation or an elongation. In the case of a substitution of one or more consecutive or non-consecutive amino acids, preference is given to substitutions in which the substituted amino acids are replaced by “equivalent” amino acids. The expression “equivalent amino acids” is intended here to denote any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding antibodies. These equivalent amino acids can be determined either on the basis of their structural homology with the amino acids for which they are substituted, or on results of comparative tests of biological activity between the various antibodies capable of being carried out.
Selon encore une autre forme d'exécution préférée, le procédé selon l'invention se caractérise par le fait que l'immunogène est le polypeptide de séquence SEQ ID N° 3, ou de séquence ayant au moins 80 % d'identité avec la séquence SEQ ID N° 3, de préférence 85 %, 90 %, 95 % ou 98 % d'identité avec la séquence comprise entre les résidus 130-230 de la séquence peptidique de ladite protéine G, ou l'un des fragments de la séquence SEQ ID N0 3 d'au moins 10 acides aminés consécutifs, de préférence d'au moins 15, 20, 25, 30 ou 50 acides aminés, capable d'induire la production d'anticorps spécifiques dirigés contre ce fragment après son administration chez un mammifère.According to yet another preferred embodiment, the method according to the invention is characterized in that the immunogen is the polypeptide of sequence SEQ ID No. 3, or of sequence having at least 80% identity with the sequence SEQ ID No. 3, preferably 85%, 90%, 95% or 98% identity with the sequence between residues 130-230 of the peptide sequence of said G protein, or one of the fragments of the sequence SEQ ID N 0 3 of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies directed against this fragment after its administration in a mammal.
D'autres immunogènes adaptés à la mise en oeuvre du procédé selon l'invention comprennent un dérivé de la protéine de surface du virus de l'hépatite A, B et C, une protéine de surface du virus de la rougeole, une protéine de surface du virus parainffuenza 3, en particulier une glycoprotéine de surface telle que hémaglutinine, neuraminidase HN et la protéine de fusion F.Other immunogens adapted to the implementation of the method according to the invention comprise a derivative of the surface protein of the hepatitis A virus, B and C, a surface protein of the measles virus, a surface protein parainffuenza 3 virus, in particular a surface glycoprotein such as hemaglutinin, neuraminidase HN and the fusion protein F.
Selon une autre forme d'exécution, la présente invention concerne un complexe immunogénique obtenu par la mise en œuvre du procédé selon l'invention.According to another embodiment, the present invention relates to an immunogenic complex obtained by the implementation of the method according to the invention.
Plus particulièrement, la présente invention a également pour objet un complexe immunogène, comprenant un immunogène, un antigène ou un haptène, caractérisé en ce que ledit immunogène est associé à un peptide support de moins de 10 acides aminés
comprenant au moins le fragment peptidique de 3 résidus d'acide aminé de séquence SEQ ID N° 2.More particularly, the present invention also relates to an immunogenic complex, comprising an immunogen, an antigen or a hapten, characterized in that said immunogen is associated with a support peptide of less than 10 amino acids comprising at least the peptide fragment of 3 amino acid residues of sequence SEQ ID No. 2.
De préférence, dans ledit complexe immunogène selon l'invention, ledit peptide support comprenant au moins le peptide codé par la SEQ ID N° 2 est constitué d'au plus 8 acides aminés, préférentiellement d'au plus 5 acides aminés, et encore mieux de 4 acides aminés.Preferably, in said immunogenic complex according to the invention, said support peptide comprising at least the peptide coded by SEQ ID No. 2 consists of at most 8 amino acids, preferably at most 5 amino acids, and even better of 4 amino acids.
Selon une forme d'exécution préférée, ledit peptide support du complexe immunogène selon l'invention consiste en le peptide codé par la SEQ ID N° 2.According to a preferred embodiment, said peptide supporting the immunogenic complex according to the invention consists of the peptide coded by SEQ ID No. 2.
Selon une forme d'exécution préférée, ledit peptide support du complexe immunogène selon l'invention est caractérisé en ce que ladite association consiste en un couplage covalent entre ledit peptide support et ledit immunogène.According to a preferred embodiment, said support peptide of the immunogenic complex according to the invention is characterized in that said association consists of a covalent coupling between said support peptide and said immunogen.
Selon une forme d'exécution préférée, ledit complexe immunogène selon l'invention est caractérisé en ce que ledit peptide support est couplé à l'extrémité N- ou C-terminale dudit immunogène lorsque ledit immunogène est un peptide, de préférence N-terminale.According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that said support peptide is coupled to the N- or C-terminal end of said immunogen when said immunogen is a peptide, preferably N-terminal.
Selon une forme d'exécution préférée, ledit complexe immunogène selon l'invention est caractérisé en ce que Pirnrnunogène est un antigène issu de bactéries, de parasites et/ou de virus.According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that Pirnrnunogène is an antigen derived from bacteria, parasites and / or viruses.
Selon une forme d'exécution préférée, ledit complexe immunogène selon l'invention est caractérisé en ce que Pimmunogène est une protéine ou glycoprotéine de surface, notamment F ou G, du virus respiratoire syncytial (VRS), ou de séquence ayant au moins 80 %d'identité avec la séquence de ladite protéine F ou G, de préférence 85 %,According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that the immunogen is a surface protein or glycoprotein, in particular F or G, of the respiratory syncytial virus (RSV), or of sequence having at least 80% identity with the sequence of said protein F or G, preferably 85%,
90 %, 95 % ou 98 % d'identité avec la séquence de ladite protéine F ou G, ou l'un de leurs fragments d'au moins 10 acides aminés consécutifs, de préférence d'au moins 15, 20, 25, 30 ou 50 acides aminés, capable d'induire la production d'anticorps spécifiques dirigés contre ce fragment après son administration chez un mammifère.90%, 95% or 98% identity with the sequence of said protein F or G, or one of their fragments of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies directed against this fragment after its administration in a mammal.
Selon une forme d'exécution préférée, ledit complexe immunogène selon l'invention est caractérisé en ce que l'immunogène est la protéine G du VRS humain de type A ou B, la protéine G du VRS bovin. Selon une forme d'exécution préférée, ledit complexe immunogène selon l'invention est caractérisé en ce que l'immunogène est le polypeptide de séquence
comprise entre les résidus 130-230 de la protéine G du VRS, extrémités comprises, ou de séquence présentant au moins 80 % d'identité avec ladite séquence 130-230, ou l'un des fragments d'au moins 10 acides aminés de ladite séquence 130-230 de la protéine G du VRS. De préférence l'immunogène dudit complexe immunogène selon l'invention est le polypeptide de séquence SEQ ID N° 3.According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that the immunogen is protein G of human RSV type A or B, protein G of bovine RSV. According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that the immunogen is the polypeptide of sequence between residues 130-230 of protein G of RSV, ends included, or of sequence having at least 80% identity with said sequence 130-230, or one of the fragments of at least 10 amino acids of said sequence 130-230 of protein G of RSV. Preferably the immunogen of said immunogenic complex according to the invention is the polypeptide of sequence SEQ ID No. 3.
Selon encore une autre forme d'exécution préférée, le complexe selon l'invention est le complexe MEFG2Na de séquence SEQ ID N° 4, ou un complexe immunogène analogue dont la séquence présente en position 1 à 3 la séquence MEF de séquence SEQ ID N° 2 suivie :According to yet another preferred embodiment, the complex according to the invention is the MEFG2Na complex of sequence SEQ ID No. 4, or an analogous immunogenic complex whose sequence has in position 1 to 3 the MEF sequence of sequence SEQ ID N ° 2 followed:
- soit d'une séquence ayant au moins 80 % d'identité avec la séquence SEQ ID N° 3, de préférence 85 %, 90 %, 95 % ou 98 % d'identité avec la séquence SEQ ID N° 3 ; ou- Or of a sequence having at least 80% identity with the sequence SEQ ID No. 3, preferably 85%, 90%, 95% or 98% identity with the sequence SEQ ID No. 3; or
- soit d'une séquence de l'un des fragments de la séquence SEQ ID N° 3. d'au moins 10 acides aminés consécutifs, de préférence d'au moins 15, 20, 25, 30 ou 50 acides aminés, capable d'induire la production d'anticorps spécifiques dirigés contre ce fragment après son administration chez un mammifère.- or of a sequence of one of the fragments of the sequence SEQ ID No. 3. of at least 10 consecutive amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of induce the production of specific antibodies directed against this fragment after its administration in a mammal.
Sous un autre aspect, la présente invention a pour objet un acide nucléique, de préférence isolé et/ou purifié, codant pour les complexes immunogènes selon l'invention, notamment pour le complexe immunogène MEFG2Na de séquence SEQ IDIn another aspect, the subject of the present invention is a nucleic acid, preferably isolated and / or purified, coding for the immunogenic complexes according to the invention, in particular for the immunogenic complex MEFG2Na of sequence SEQ ID
N0 4.N 0 4.
Par acide nucléique, séquence nucléique ou d'acide nucléique, polynucléotide, oligonucléotide, séquence de polynucléotide, séquence nucléotidique, termes qui seront employés indifféremment dans la présente description, on entend désigner un enchaînement précis de nucléotides, modifiés ou non, permettant de définir un fragment ou une région d'un acide nucléique, comportant ou non des nucléotides non naturels, et pouvant correspondre aussi bien à un ADN double brin, un ADN simple brin que des produits de transcription desdits ADNs.The term “nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs.
Sous encore un autre aspect, la présente invention a pour objet les complexes immunogènes selon l'invention ou les acides nucléiques codant pour les complexes immunogènes selon l'invention à titre de médicament, notamment le complexe
immunogène MEFG2Na de séquence SEQ ID N° 4 ou l'acide nucléique tel qu'un ADN ou ARN, codant pour ce complexe MEFG2Na.In yet another aspect, the subject of the present invention is the immunogenic complexes according to the invention or the nucleic acids coding for the immunogenic complexes according to the invention as a medicament, in particular the complex MEFG2Na immunogen of sequence SEQ ID No. 4 or nucleic acid such as DNA or RNA, coding for this MEFG2Na complex.
Les compositions pharmaceutiques comprenant les complexes immunogènes selon l'invention ou tels que définis précédemment, ou un acide nucléique, ARN ou ADN, codant pour de tels complexes immunogènes, associés avec des excipients physiologiquement acceptables font également partie de l'invention. Ils sont particulièrement adaptés à la préparation d'un vaccin.Pharmaceutical compositions comprising the immunogenic complexes according to the invention or as defined above, or a nucleic acid, RNA or DNA, coding for such immunogenic complexes, associated with physiologically acceptable excipients also form part of the invention. They are particularly suitable for the preparation of a vaccine.
L'immunisation pourra être obtenue par l'administration dudit polynucléotide codant pour les complexes immunogènes tels que définis précédemment, seul ou par l'intermédiaire d'un vecteur viral comprenant un tel polynucléotide. On peut également utiliser une cellule hôte, notamment une bactérie inactivée, transformée avec un tel polynucléotide selon l'invention.Immunization may be obtained by the administration of said polynucleotide encoding the immunogenic complexes as defined above, alone or through a viral vector comprising such a polynucleotide. It is also possible to use a host cell, in particular an inactivated bacterium, transformed with such a polynucleotide according to the invention.
La présente invention vise également l'utilisation d'un complexe immunogène selon l'invention, dans lequel complexe ledit immunogène est une protéine ou un peptide dérivé de la protéine G ou F du VRS telle que définie précédemment, notamment le complexe MEFG2Na ou l'un de ses analogues selon l'invention, ou un acide nucléique selon l'invention codant pour ledit complexe immunogène, pour la préparation d'une composition pharmaceutique destinée à la prévention ou au traitement des infections respiratoires liées au VRS. Les avantages de la présente invention seront mis en évidence à la lumière des exemples et des figures ci-après dans lesquels :The present invention also relates to the use of an immunogenic complex according to the invention, in which complex said immunogen is a protein or a peptide derived from the G or F protein of RSV as defined above, in particular the MEFG2Na complex or the one of its analogs according to the invention, or a nucleic acid according to the invention encoding said immunogenic complex, for the preparation of a pharmaceutical composition intended for the prevention or treatment of respiratory infections linked to RSV. The advantages of the present invention will be demonstrated in the light of the examples and figures below in which:
- la figure 1 représente le taux d'IgG anti-RSV-A chez des souris immunisées avec BBG2Na ou MEFG2Na ;- Figure 1 shows the level of anti-RSV-A IgG in mice immunized with BBG2Na or MEFG2Na;
- la figure 2 représente également, selon une autre présentation, le taux d'IgG anti-RSV-A chez des souris immunisées avec BBG2Na ou MEFG2Na après 2 immunisations ;- Figure 2 also shows, according to another presentation, the level of anti-RSV-A IgG in mice immunized with BBG2Na or MEFG2Na after 2 immunizations;
- la figure 3 représente le taux d'IgG anti-G2Na chez des souris immunisées avec BBG2Na ou MEFG2Na ; et- Figure 3 shows the level of anti-G2Na IgG in mice immunized with BBG2Na or MEFG2Na; and
- la figure 4 représente également, selon une autre présentation, le taux d'IgG anti-G2Na chez des souris immunisées avec BBG2Na ou MEFG2Na.
Exemple 1 : Comparaison des activités in-vivo induites par l'utilisation de la protéine porteuse BB ou du peptide support MEF- Figure 4 also shows, according to another presentation, the level of anti-G2Na IgG in mice immunized with BBG2Na or MEFG2Na. Example 1 Comparison of the In Vivo Activities Induced by the Use of the BB Carrier Protein or of the MEF Support Peptide
Des souris BALB/c femelles IOPS âgées de 8 semaines sont infectées par voie nasale avec du RSV-A souche Long (105 pfu) à J-20. A JO, après confirmation de la séroconversion pour le RSV-A, les souris reçoivent une seule injection intra-musculaire de 20 μg de BBG2Na adsorbé sur Adju-Phos (soit 6 μg équivalent G2Na) ou 6 μg de MEFG2Na adsorbé sur Adju-Phos. Le taux d'IgG anti-RSV-A (antigène viral purifié) et d'anti-MEFG2Na est suivi par ELISA.8-week-old female IOPS BALB / c mice are infected nasally with RSV-A Long strain (10 5 pfu) at D-20. On the OJ, after confirmation of seroconversion for RSV-A, the mice receive a single intramuscular injection of 20 μg of BBG2Na adsorbed on Adju-Phos (i.e. 6 μg equivalent G2Na) or 6 μg of MEFG2Na adsorbed on Adju-Phos . The level of anti-RSV-A IgG (purified viral antigen) and anti-MEFG2Na is monitored by ELISA.
Les figures 1 et 2 montrent qu'il n'y a pas de différence significative entre le taux d'IgG anti-RSV-A déclenché par 6 μg de MEFG2Na ou 20 μg de BBG2Na, et ceci en aucun point de la cinétique. Il en est de même pour le taux d'IgG anti-G2Na (figures 3 et 4).Figures 1 and 2 show that there is no significant difference between the level of anti-RSV-A IgG triggered by 6 μg of MEFG2Na or 20 μg of BBG2Na, and this at no point in the kinetics. The same is true for the anti-G2Na IgG level (Figures 3 and 4).
Exemple 2 : Préparation des complexes BBG2Na et MEFG2Na Préparation de BBG2Na : La protéine BBG2Na est produite en utilisant Escherichia coli RV308 comme cellule hôte et un plasmide où la transcription du gène d'intérêt est sous le contrôle du promoteur trytophane. L'étape de fermentation est un procédé de type batch avec un milieu de culture synthétique semi-défini et du glycérol comme source de carbone et d'énergie. Deux étapes de culture sont nécessaires pour préparer l'inoculum qui est utilisé pour ensemencer le fermenteur de production. Dans ce fermenteur, les microorganismes sont cultivés jusqu'à une densité optique à 620 nm de 50, puis l'expression est induite par addition d'un analogue du tryptophane (IAA). La culture est poursuivie jusqu'à ce que la pression partielle d'O2 dans le fermenteur remonte subitement, ce qui signale l'épuisement de la source de carbone. A ce stade la densité cellulaire moyenne est de 40 g de cellules sèches/litre avec un taux d'expression deExample 2: Preparation of BBG2Na and MEFG2Na complexes Preparation of BBG2Na: The BBG2Na protein is produced using Escherichia coli RV308 as host cell and a plasmid where the transcription of the gene of interest is under the control of the trytophane promoter. The fermentation stage is a batch type process with a semi-defined synthetic culture medium and glycerol as a source of carbon and energy. Two culture steps are necessary to prepare the inoculum which is used to seed the production fermenter. In this fermenter, the microorganisms are cultured up to an optical density at 620 nm of 50, then expression is induced by the addition of a tryptophan analog (IAA). The culture is continued until the partial pressure of O 2 in the fermenter suddenly rises, which signals the exhaustion of the carbon source. At this stage, the average cell density is 40 g of dry cells / liter with an expression rate of
9,5 %, soit une productivité de 3,8 g de BBG2Na/litre de culture. La culture est refroidie à +40C, les micro-organismes sont récupérés par centrifugation et congelés à -15/-25°C.9.5%, i.e. a productivity of 3.8 g of BBG2Na / liter of culture. The culture is cooled to +4 ° C., the microorganisms are recovered by centrifugation and frozen at -15 / -25 ° C.
L'extraction de BBG2Na nécessite une solubilisation du culot de microorganismes décongelé avec un tampon contenant de la guanidine, HCl et du 1,4- dithiothreitol (DTT) pour réduire les ponts disulfures. La renaturation de la protéine et l'oxydation des ponts disulfures sont obtenues par dilution de la suspension dénaturée et
agitation à température ambiante pendant une nuit dans un réacteur ouvert. La suspension contenant la protéine renaturée est clarifiée par centrifugation puis filtrée. Ensuite, du PEG 6000 est ajouté au filtrat et le précipité qui en résulte est récupéré par centrifugation. Le précipité contenant BBG2Na est solubilisé de nouveau dans un tampon contenant de l'urée. L'extrait obtenu est filtré sur un support 0,22 μm et conservé à -15/-25°C.The extraction of BBG2Na requires solubilization of the pellet of thawed microorganisms with a buffer containing guanidine, HCl and 1,4-dithiothreitol (DTT) to reduce the disulfide bridges. The renaturation of the protein and the oxidation of the disulfide bridges are obtained by diluting the denatured suspension and stirring at room temperature overnight in an open reactor. The suspension containing the renatured protein is clarified by centrifugation and then filtered. Then, PEG 6000 is added to the filtrate and the resulting precipitate is recovered by centrifugation. The precipitate containing BBG2Na is solubilized again in a buffer containing urea. The extract obtained is filtered through a 0.22 μm support and stored at -15 / -25 ° C.
La purification de BBG2Na à partir de l'extrait décongelé comporte cinq étapes qui sont : (1) une chromatographie d'échange de cations sur une colonne de SP- Sepharose fast flow, (2) une chromatographie d'interaction hydrophobe sur une colonne de methyl Macroprep, (3) une gel filtration sur une colonne de Superdex S200, (4) une chromatographie d'échange d'anions sur une colonne de DEAE-Sepharose et finalement (5) une étape de dessalage sur une colonne de Sephadex G25. La solution de protéine purifiée est filtrée stérilement et répartie dans des poches stériles et apyrogènes. Préparation de MEFG2Na : La protéine MEFG2Na est produite en utilisant Escherichia coli ICONE 200 comme cellule hôte et un plasmide ou la transcription du gène d'intérêt est sous le contrôle du promoteur trytophane. E. coli ICONE 200 est un mutant de E. coli RV308 qui a été développé pour améliorer le contrôle de l'expression pendant la phase de croissance. L'étape de fermentation est un procédé de type fed-batch avec un milieu de culture chimiquement défini et du glycérol comme source de carbone et d'énergie. Deux étapes de culture sont nécessaires pour préparer l'inoculum qui est utilisé pour ensemencer le fermenteur de production. Dans ce fermenteur, les microorganismes sont cultivés jusqu'à une densité optique à 620 nm de 110, puis l'expression est induite par addition d'un analogue du tryptophane (IAA). La culture est poursuivie jusqu'à ce que la pression partielle d'O2 dans le fermenteur remonte subitement, ce qui signale l'épuisement de la source de carbone. A ce stade la densité cellulaire moyenne est de 56 g de cellules sèches/litre avec un taux d'expression de 5,4 %, soit une productivité de 3 g de MEFG2Na/litre de culture. La culture est refroidie à +4°C, les micro-organismes sont récupérés par centrifugation et congelés à -15/-25°C. L'extraction de MEFG2Na nécessite une solubilisation du culot de micro¬ organismes décongelé avec un tampon contenant de la guanidine, HCl. La suspension
contenant la protéine dénaturée est clarifiée par centrifugation puis filtrée. La guanidine étant incompatible avec l'étape de purification subséquente une étape de concentration dialyse, sur un support d'ultrafiltration en polyéthersulfone avec un seuil de coupure à 10 kDa, est mise en place pour effectuer le changement de tampon. L'extrait obtenu- est filtré sur un support 0,22 μm et purifié dans la foulée.The purification of BBG2Na from the thawed extract comprises five stages which are: (1) cation exchange chromatography on a column of SP-Sepharose fast flow, (2) hydrophobic interaction chromatography on a column of methyl Macroprep, (3) a gel filtration on a column of Superdex S200, (4) an anion exchange chromatography on a column of DEAE-Sepharose and finally (5) a desalting step on a column of Sephadex G25. The purified protein solution is sterile filtered and distributed in sterile and pyrogen-free bags. Preparation of MEFG2Na: The MEFG2Na protein is produced using Escherichia coli ICONE 200 as host cell and a plasmid where the transcription of the gene of interest is under the control of the trytophan promoter. E. coli ICONE 200 is a mutant of E. coli RV308 which was developed to improve expression control during the growth phase. The fermentation step is a fed-batch type process with a chemically defined culture medium and glycerol as a source of carbon and energy. Two culture steps are necessary to prepare the inoculum which is used to seed the production fermenter. In this fermenter, the microorganisms are grown up to an optical density at 620 nm of 110, then expression is induced by the addition of a tryptophan analog (IAA). The culture is continued until the partial pressure of O 2 in the fermenter suddenly rises, which signals the exhaustion of the carbon source. At this stage, the average cell density is 56 g of dry cells / liter with an expression rate of 5.4%, ie a productivity of 3 g of MEFG2Na / liter of culture. The culture is cooled to + 4 ° C., the microorganisms are recovered by centrifugation and frozen at -15 / -25 ° C. The extraction of MEFG2Na requires solubilization of the pellet of thawed micro¬ organisms with a buffer containing guanidine, HCl. Suspension containing the denatured protein is clarified by centrifugation and then filtered. Since guanidine is incompatible with the subsequent purification step, a dialysis concentration step, on a polyethersulfone ultrafiltration support with a cutoff threshold of 10 kDa, is put in place to effect the change of buffer. The extract obtained is filtered on a 0.22 μm support and purified in stride.
La purification de MEFG2Na comporte trois étapes qui sont : (1) une chromatographie d'échange de cations sur une colonne de Fractogel EMD SE Hicap, (2) une gel filtration sur une colonne de Superdex 75 Prep Grade, (3) une chromatographie d'échange d'anions sur une colonne de DEAE Sépharose Fast Flow. Le vrac de protéine purifiée est filtré stérilement et réparti dans des poches stériles et apyrogènes. Rendements d'expression :The purification of MEFG2Na comprises three stages which are: (1) cation exchange chromatography on a column of Fractogel EMD SE Hicap, (2) gel filtration on a column of Superdex 75 Prep Grade, (3) chromatography of exchange of anions on a column of DEAE Sepharose Fast Flow. The bulk of purified protein is sterile filtered and distributed in sterile and pyrogen-free bags. Expression yields:
Les données d'expression de MEFG2Na et de BBG2Na sont regroupées dans le tableau 1 suivant. Tableau 1 : Quantité de protéine MEFG2Na et de BBG2Na obtenue exprimée en Mol/ 100g de cellules sèchesThe MEFG2Na and BBG2Na expression data are collated in Table 1 below. Table 1: Quantity of MEFG2Na protein and of BBG2Na obtained expressed in Mol / 100g of dry cells
II apparaît que le taux d'expression du complexe MEFG2Na est supérieur d'environ deux fois au taux d'expression du complexe BBG2Na. Bien que la présente description, ainsi que les exemples, sont basés uniquement sur l'antigène G2Na, il faut comprendre que tout immunogène peut également être couplé au peptide support objet de la présente invention.
It appears that the level of expression of the MEFG2Na complex is approximately twice as high as the level of expression of the BBG2Na complex. Although the present description, as well as the examples, are based solely on the G2Na antigen, it should be understood that any immunogen can also be coupled to the support peptide object of the present invention.
Claims
1. Procédé de préparation d'un complexe immunogène dans lequel on associe un immunogène, antigène ou haptène, à un peptide support pour former ledit complexe immunogène, caractérisé en ce que ledit peptide support consiste en un peptide de moins de 10 acides aminés comprenant au moins le peptide de séquence SEQ ID N° 2.1. A method of preparing an immunogenic complex in which an immunogen, antigen or hapten, is associated with a support peptide to form said immunogenic complex, characterized in that said support peptide consists of a peptide of less than 10 amino acids comprising at minus the peptide of sequence SEQ ID No. 2.
2. Procédé selon la revendication 1, caractérisé en ce que ledit peptide support de moins de 10 acides aminés consiste en le peptide codé par la SEQ ID N° 2. 2. Method according to claim 1, characterized in that said support peptide of less than 10 amino acids consists of the peptide coded by SEQ ID No. 2.
3. Procédé selon la revendication 1 ou 2, caractérisé en ce que ladite association consiste en un couplage covalent entre ledit peptide support et ledit immunogène.3. Method according to claim 1 or 2, characterized in that said association consists of a covalent coupling between said support peptide and said immunogen.
4. Procédé selon la revendication 3, caractérisé en ce que ledit peptide support est couplé à l'extrémité N-terminale dudit immunogène lorsque ledit immunogène est un peptide.4. Method according to claim 3, characterized in that said support peptide is coupled to the N-terminal end of said immunogen when said immunogen is a peptide.
5. Procédé selon la revendication 4, caractérisé en ce que ledit couplage covalent est réalisé par la technologie de l' ADN recombinant.5. Method according to claim 4, characterized in that said covalent coupling is carried out by the technology of recombinant DNA.
6. Procédé selon la revendication 3 ou 4, caractérisé en ce que ledit couplage covalent est réalisé par voie chimique. 6. Method according to claim 3 or 4, characterized in that said covalent coupling is carried out chemically.
7. Procédé selon l'une des revendications 1 à 6, caractérisé en ce que7. Method according to one of claims 1 to 6, characterized in that
Pimmunogène est un antigène issu de bactéries, de parasites et/ou de virus.The immunogen is an antigen from bacteria, parasites and / or viruses.
8. Procédé selon la revendication 7, caractérisé en ce que l'immunogène est une protéine ou glycoprotéine de surface du virus respiratoire syncytial (VRS), une protéine de séquence présentant au moins 80 % d'identité avec la séquence de ladite protéine de surface du VRS ou l'un des fragments d'au moins 10 acides aminés consécutifs de ladite protéine de surface du VRS, ladite protéine de séquence présentant au moins 80 % d'identité ou ledit fragment étant capable d'induire la production d'anticorps spécifiques dirigés contre cette protéine ou cedit fragment après son administration chez un mammifère. 8. Method according to claim 7, characterized in that the immunogen is a surface protein or glycoprotein of the respiratory syncytial virus (RSV), a protein of sequence having at least 80% identity with the sequence of said surface protein RSV or one of the fragments of at least 10 consecutive amino acids of said RSV surface protein, said sequence protein having at least 80% identity or said fragment being capable of inducing the production of specific antibodies directed against this protein or said fragment after its administration in a mammal.
9. Procédé selon la revendication 8, caractérisé en ce que l'immunogène est la protéine G du VRS humain de type A ou B, la protéine G du VRS bovin, une protéine
de séquence présentant au moins 80 % d'identité avec la séquence de ladite protéine G ou l'un des fragments de ladite protéine G d'au moins 10 acides aminés.9. Method according to claim 8, characterized in that the immunogen is protein G of human RSV type A or B, protein G of bovine RSV, a protein of sequence having at least 80% identity with the sequence of said G protein or one of the fragments of said G protein of at least 10 amino acids.
10. Procédé selon la revendication 9, caractérisé en ce que l'immunogène est le polypeptide de séquence comprise entre les résidus 130-230 de la protéine G du VRS, extrémités comprises, ou de séquence présentant au moins 80 % d'identité avec ladite séquence 130-230, ou l'un des fragments d'au moins 10 acides aminés de ladite protéine G.10. Method according to claim 9, characterized in that the immunogen is the polypeptide of sequence between the residues 130-230 of protein G of the RSV, ends included, or of sequence having at least 80% identity with said sequence 130-230, or one of the fragments of at least 10 amino acids of said protein G.
11. Procédé selon la revendication 10, caractérisé en ce que l'immunogène est le polypeptide de séquence SEQ ID N° 3. 11. Method according to claim 10, characterized in that the immunogen is the polypeptide of sequence SEQ ID No. 3.
12. Complexe immunogène obtenu par la mise en œuvre du procédé selon l'une des revendications 8 à 11.12. Immunogenic complex obtained by implementing the method according to one of claims 8 to 11.
13. Complexe immunogène, comprenant un immunogène, antigène ou haptène, associé à un peptide support, caractérisé en ce que :13. Immunogenic complex, comprising an immunogen, antigen or hapten, associated with a support peptide, characterized in that:
- ledit immunogène est associé, de préférence couplé par liaison covalente, à un peptide support de moins de 10 acides aminés comprenant au moins le peptide de séquence SEQ ID N° 2 ; et en ce que :- Said immunogen is associated, preferably coupled by covalent bond, with a support peptide of less than 10 amino acids comprising at least the peptide of sequence SEQ ID No. 2; and in that :
- l'immunogène est une protéine ou glycoprotéine de surface, notamment F ou G, du virus respiratoire syncytial (VRS), ou de séquence ayant au moins 80 % d'identité avec la séquence de ladite protéine de surface du VRS, capable d'induire la production d'anticorps spécifiques dirigés contre cette dite protéine de séquence ayant au moins 80 % d'identité après leur administration chez un mammifère..the immunogen is a surface protein or glycoprotein, in particular F or G, of the respiratory syncytial virus (RSV), or of sequence having at least 80% identity with the sequence of said surface protein of the RSV, capable of induce the production of specific antibodies directed against this so-called sequence protein having at least 80% identity after their administration in a mammal.
14. Complexe selon la revendication 13, caractérisé en ce que ledit peptide support est le peptide de séquence SEQ ID N° 2.14. Complex according to claim 13, characterized in that said support peptide is the peptide of sequence SEQ ID No. 2.
15. Complexe selon la revendication 12 ou 13, caractérisé en ce qu'il s'agit du complexe MEFG2Na de séquence SEQ ID N° 4, ou d'un complexe immunogène dont la séquence présente en position 1 à 3 la séquence SEQ ID N° 2 suivie :15. Complex according to claim 12 or 13, characterized in that it is the MEFG2Na complex of sequence SEQ ID No. 4, or an immunogenic complex whose sequence has in position 1 to 3 the sequence SEQ ID N ° 2 followed:
- soit d'une séquence ayant au moins 80 % d'identité avec la séquence SEQ ID N° 3, de préférence 85 %, 90 %, 95 % ou 98 % d'identité avec la séquence SEQ ID N° 3.- Or of a sequence having at least 80% identity with the sequence SEQ ID No. 3, preferably 85%, 90%, 95% or 98% identity with the sequence SEQ ID No. 3.
16. Complexe selon la revendication 15, de séquence SEQ ID N° 4. 16. Complex according to claim 15, of sequence SEQ ID No. 4.
17. Acide nucléique codant pour un complexe immunogène selon l'une des revendications 13 à 16.
17. Nucleic acid coding for an immunogenic complex according to one of claims 13 to 16.
18. Acide nucléique selon la revendication 17 codant pour le complexe immunogène de séquence SEQ ID N° 4.18. Nucleic acid according to claim 17 coding for the immunogenic complex of sequence SEQ ID No. 4.
19. Complexe selon l'une des revendications 12 à 16 ou acide nucléique selon la revendication 17 ou 18, à titre de médicament.19. Complex according to one of claims 12 to 16 or nucleic acid according to claim 17 or 18, as a medicament.
20. Utilisation d'un complexe immunogène selon l'une des revendications 12 à 16 ou d'un acide nucléique selon la revendication 17 ou 18, pour la préparation d'une composition pharmaceutique destinée au traitement ou à la prévention des infections respiratoires liés au VRS.
20. Use of an immunogenic complex according to one of claims 12 to 16 or a nucleic acid according to claim 17 or 18, for the preparation of a pharmaceutical composition intended for the treatment or prevention of respiratory infections linked to VRS.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0408175A FR2873378A1 (en) | 2004-07-23 | 2004-07-23 | IMMUNOGENIC COMPLEXES, PROCESS FOR THEIR PREPARATION AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS |
| PCT/FR2005/001913 WO2006018527A1 (en) | 2004-07-23 | 2005-07-25 | Immunogenic complexes, preparation method thereof and use of same in pharmaceutical compositions |
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| EP (1) | EP1776379A1 (en) |
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| AU (1) | AU2005273779A1 (en) |
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| FR (1) | FR2873378A1 (en) |
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| EP2324062A4 (en) * | 2008-07-18 | 2012-06-06 | Id Biomedical Corp Quebec | Chimeric respiratory syncytial virus polypeptide antigens |
| US8889146B2 (en) | 2009-06-24 | 2014-11-18 | Glaxosmithkline Biologicals, Sa | Vaccine |
| PE20121541A1 (en) | 2009-06-24 | 2012-12-21 | Glaxosmithkline Biolog Sa | RECOMBINANT RESPIRATORY SYNCIZAL VIRUS ANTIGENS |
| SG178026A1 (en) | 2009-07-15 | 2012-03-29 | Novartis Ag | Rsv f protein compositions and methods for making same |
| BR112012008338A2 (en) | 2009-09-10 | 2019-09-24 | Novartis Ag | combination of vaccines against respiratory tract diseases. |
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| FR2798292A1 (en) * | 1999-09-09 | 2001-03-16 | Pf Medicament | Use of quaternary aliphatic ammonium salt and immunogen or antigen to combat respiratory syncytial virus infections |
| FR2827606A1 (en) * | 2001-07-20 | 2003-01-24 | Pf Medicament | New peptide derived from diphtheria anatoxin, useful as carrier in vaccines, lacks at least one Cys residue, also related nucleic acids |
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| NO20071030L (en) | 2007-04-23 |
| CA2574340A1 (en) | 2006-02-23 |
| WO2006018527A1 (en) | 2006-02-23 |
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| AU2005273779A1 (en) | 2006-02-23 |
| KR20070058457A (en) | 2007-06-08 |
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