HK1100949A - Immunogenic complexes, preparation method thereof and use of same in pharmaceutical compositions - Google Patents

Description

Immunogenic complexes, method for the preparation thereof and use thereof in pharmaceutical compositions

The present invention relates to methods for increasing the immunogenicity of immunogens, antigens or haptens by coupling with small support peptides. More specifically, the invention relates to a method for preparing an immunogenic complex, as well as to a complex obtainable by such a method, and to the use of said complex as a medicament to increase the immunogenicity of an immunogen. For example, the invention encompasses a support peptide coupled to a peptide from the Respiratory Syncytial Virus (RSV) G protein, and its use as a vaccine for the treatment of RSV-related respiratory infections.

The immune system is a network of interacting humoral and cellular components that allows the host to distinguish between self and non-self molecules to remove the latter, as well as pathogens. To this end, the immune system has developed two mechanisms that act synergistically, namely innate immunity and adaptive immunity.

Natural immunity includes physical barriers (skin, mucosa, etc.), cells activated or produced in response to challenge (monocytes/macrophages, granulocytes, NK cells, etc.), and soluble factors (complement, cytokines, acute phase proteins, etc.). The innate immune response is rapid, but it is neither specific nor memorized.

The cellular mediators of acquired immunity are T and B lymphocytes. In particular, the latter produce immunoglobulins through their interaction. The immune response of acquired immunity is specific, adaptable and can be remembered, in contrast to the natural immune response. In fact, the initial invasion of antigen into naive (n-gapped ive) organisms results in an immune response, termed the primary response, during which long-lived lymphocytes (T and B), termed memory cells, expand. By these cells, during the second penetration of the same antigen, the immune response, known as the secondary reaction, will be faster and stronger. To mount a primary response, antigen must first be captured and prepared by antigen presenting cells for presentation to T lymphocytes.

The purpose of a vaccine is to protect the host by preventing or limiting pathogen invasion. All vaccines currently marketed achieve this effect by eliciting antibodies to be produced.

When the vaccination antigen alone cannot elicit an immune response, or if an immune response is generated but is too weak, its physical association with a so-called carrier protein having T epitopes capable of interacting with T lymphocytes is capable of eliciting the desired response. The most commonly known vaccine carrier proteins are diphtheria and tetanus toxoids.

Among these carrier proteins, mention may also be made of the so-called "BB" protein fragment of the streptococcal G protein, which is capable of binding to albumin and is a fragment corresponding to residues 24 to 242 of the sequence of SEQ ID NO 1. This protein is capable of eliciting an earlier and stronger primary antibody response to the vaccinating antigen to which it binds (Libon et al, Vaccine, 17 (5): 406-41, 1999). In this context, reference is also made to international patent application WO 96/14416.

It is an object of the present invention to provide an alternative to carrier proteins which will remedy all the disadvantages associated with such carrier proteins as described in the following description. More specifically, the invention enables limiting the side effects associated with the presence of relatively large carrier proteins while allowing high yields to be achieved.

For clarity, the advantages of the present invention are demonstrated by comparison with the existing carrier protein, namely BB carrier protein.

Quite unexpectedly, and contrary to the prevailing knowledge accepted by the skilled person, the inventors have demonstrated the use of alternatives to carrier proteins. More specifically, the inventors have characterized a method for increasing the immunogenicity of immunogens based on the identification of very small and therefore non-immunogenic peptides, hereinafter referred to as supporting peptides, which facilitate their synthesis and/or the synthesis of the immunogen-supporting peptide complexes in which they are involved.

To this end, the invention relates to a method for preparing an immunogenic complex, wherein an immunogen, an antigen or a hapten is coupled to a support peptide to form said immunogenic complex, wherein said support peptide consists of a peptide of less than 10 amino acids comprising at least the 3 amino acid residue peptide fragment of the sequence SEQ ID NO2 (Met-Glu-Phe).

The term "immunogen" includes any substance that is capable of eliciting an immune response. By way of non-limiting example, the immunogen is preferably a protein, glycoprotein, lipopeptide or any immunogenic compound comprising at least 5 amino acids, preferably at least 10, 15, 20, 25, 30 or 50 amino acids in its structure, which compound is capable of eliciting an immune response, in particular of inducing the production of specific antibodies against said peptide, upon administration to a mammal.

In the present specification, the terms "polypeptide", "polypeptide sequence", "peptide" and "protein" are interchangeable.

For the above description, it should be clearly understood that the expression "support peptide" is not equivalent to "carrier protein". Indeed, the carrier protein is characterized by its large size (218 amino acids for BB protein), and most importantly by the presence of T epitopes capable of binding to T antigen receptors on the surface of T lymphocytes. The support peptide according to the invention differs from the carrier protein by the fact that the support peptide is much smaller (less than 10 amino acids) and by the fact that the support peptide does not exhibit a T epitope.

According to a first advantageous aspect, the method according to the invention makes it possible to produce immune complexes that increase the immunogenicity of the immunogen, for which production is easier or for which the yield is higher. In fact, the complexes comprising the support peptides according to the invention are much smaller than the complexes comprising the carrier proteins of the prior art, which are easier to prepare by peptide/chemical synthesis or any other technique known to the person skilled in the art.

According to a second advantageous aspect, the immunogenic complex according to the invention makes it possible to eliminate, at least limit, the adverse effects associated with the particular nature of the carrier protein. It is accepted by those skilled in the art that a relatively large carrier protein, such as BB, has a higher probability of being the cause of an undesirable immune response. For example, for tetanus toxoid, pre-sensitization of the host to this carrier protein has been shown to prevent an antibody response against antigens bound to tetanus toxoid during vaccination with the conjugate (Kaliyaperumal et al, eur.j.immunol., 25 (12): 3375-80, 1995). This phenomenon is called epitope suppression.

Thus, it is clear from the present description that the present invention provides an advantageous alternative to the use of carrier proteins. Indeed, due to its small size, the support protein has no, or very little, chance as a source of side effects or undesirable effects.

According to a preferred embodiment of the invention, the support peptide of less than 10 amino acids comprises at least the peptide encoded by SEQ ID NO2 and consists of at most 8 amino acids, preferably at most 5 amino acids, more preferably 4 amino acids.

According to another preferred embodiment, the support peptide of less than 10 amino acids according to the invention consists of the peptide of the sequence of SEQ ID NO 2.

The conjugation between the support peptide and the immunogen may be performed by coupling techniques known to those skilled in the art that preserve the integrity and immunogenicity of the immunogen. More particularly, the method according to the invention is characterized in that said association consists of covalent coupling. The term "covalent coupling" includes chemical coupling or protein fusion by the so-called recombinant DNA technique, wherein the fusion protein is obtained by a host cell (eukaryotic or prokaryotic) transformed with the nucleic acid after translation of the nucleic acid encoding the fusion protein (immunogenic complex).

When the immunogen is a peptide, the support peptide may be coupled at the N-terminus or C-terminus of the immunogen. Preferably, the support peptide is coupled to the N-terminus of the immunogen.

Complexes between the support peptide and the compounds sought to enhance immunogenicity can be produced by recombinant DNA techniques, particularly by inserting or fusing DNA encoding the immunogen into a DNA molecule encoding the support.

According to another embodiment, the covalent coupling between the support peptide and the immunogen is carried out by chemical routes known to the person skilled in the art.

As an object, the present invention also provides a method in which the immunogenic complex is obtained by genetic recombination (recombinant protein) using a nucleic acid derived from a DNA molecule encoding a supporting peptide fused (or inserted) to a DNA encoding an immunogen, with a promoter if necessary.

In this method, vectors comprising such fusion nucleic acids, in particular DNA vectors originating from plasmids, phages, viruses and/or cosmids and which fusion nucleic acids encode said complexes, which are capable of integrating into the genome of the host cell and thereby of being expressed therein, can be used.

Thus in one embodiment thereof, the method according to the invention comprises the step of producing said complex in a host cell by genetic engineering.

The host cell may be prokaryotic and is in particular selected from the group consisting of E.coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus; it may also be a yeast.

According to another aspect, the host cell is a eukaryotic cell, such as a mammalian cell or an insect cell (Sf 9).

The fusion nucleic acid encoding the immunogenic complex can be introduced into a host cell, inter alia, by a viral vector.

The immunogen used is preferably derived from a bacterium, parasite, virus or tumor-associated antigen, such as an antigen associated with melanoma or derived from β -hCG.

The method according to the invention is particularly suitable for surface polypeptides of pathogens. When the polypeptide is expressed as a fusion protein by recombinant DNA techniques, the fusion protein is advantageously expressed, anchored and exposed to the surface of the host cell membrane. The nucleic acid molecules used are capable of directing antigen synthesis in a host cell.

The molecule comprises a promoter sequence, a functionally linked secretion signal sequence and a sequence encoding a membrane anchoring region, all of which can be adapted by the person skilled in the art.

The immunogen may in particular be derived from human RSV of type a or B or bovine RSV surface glycoproteins, in particular selected from the F and G proteins.

Particularly advantageous results are obtained with human RSV G protein, subgroup A or B, or bovine RSV.

In a preferred form, the immunogen consists of a polypeptide encoded by a sequence between residues 130-230 of the RSV G protein peptide sequence, or by any sequence having at least 80% identity to said peptide sequence, wherein preferably 85%, 90%, 95% or 98% identity to the sequence between residues 130-230 of said G protein peptide sequence, or a fragment thereof of at least 10 contiguous amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, capable of inducing the production of specific antibodies against said fragment upon administration thereof to a mammal.

In the present invention, the "percent identity" or "percent homology" (the two expressions are interchangeable in this specification) between two nucleic acid or amino acid sequences denotes the percentage of nucleotides or amino acid residues that are identical between the two sequences that are compared after optimal alignment (optimal matching), which percentage is purely statistical and the differences between the two sequences are randomly distributed over their length. Sequence comparisons between two nucleic acid or amino acid sequences are typically performed by comparing the sequences after they have been optimally aligned, either by segments or "comparison windows". Optimal alignment of sequences for comparison can be performed manually or by the Smith-Waterman local homology algorithm (1981) [ ad. 482], Needleman-Wunsch local homology algorithm (1970) [ J.mol.biol.48: 443], Pearson and Lipman similarity search method (1988) [ proc.natl.acad.sci.usa 85: 2444 or by Computer software (Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, GAP in Wis, BESTFIT, FASTA and TFASTA, or BLAST N or BLASTP comparison software) using these algorithms.

The percent identity between two nucleic acid or amino acid sequences is determined by optimally comparing the two aligned sequences, wherein the nucleic acid or amino acid sequence to be compared may include additions or deletions compared to the reference sequence for optimal matching between the two sequences. Percent identity was calculated as follows: percent identity between two sequences is obtained by determining the number of identical positions having the same nucleotide or amino acid between the two sequences, dividing the number of identical positions by the total number of positions in the window of comparison, and multiplying the result by 100.

For example, the website address may behttp://www.ncbi.nlm.nih.gov/gorf/b12.htmlThe BLAST programs, "BLAST 2 sequences" (Tatusova et al, "BLAST 2 sequences-a new tool for matching protein and nucleotide sequences," FEMSMicrocobiol Lett.174: 247-.

For amino acid sequences having at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, sequences having certain modifications compared to the reference sequence, in particular a deletion, addition or substitution, truncation or extension of at least one amino acid, are preferred. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitution in which the substituted amino acid is replaced by an "equivalent" amino acid is preferred. The expression "equivalent amino acid" denotes here any amino acid which may be substituted as one of the amino acids of the basic structure, without substantially altering the biological activity of the corresponding antibody. These equivalent amino acids can be determined based on their structural homology with the amino acids to be substituted by them, or based on the results of comparative tests of biological activity between the various antibodies that may be produced.

According to another preferred embodiment, the method according to the invention is characterized by the fact that: the immunogen is a polypeptide of the sequence of SEQ ID NO3, or a sequence having at least 80% identity to the sequence of SEQ ID NO3, preferably a sequence having 85%, 90%, 95% or 98% identity to the sequence between residues 130 and 230 of the peptide sequence of said G protein, or a fragment of at least 10 consecutive amino acids of the sequence of SEQ ID NO3, preferably at least 15, 20, 25, 30 or 50 amino acids, which is capable of inducing the production of specific antibodies against said fragment upon administration to a mammal.

Other immunogens suitable for carrying out the method of the invention include derivatives of hepatitis A, B and C virus surface proteins, measles virus surface proteins, parainfluenza virus surface proteins, especially surface glycoproteins such as hemagglutinin, neuraminidase, hemagglutinin-neuraminidase (HN) and fusion (F) proteins.

According to another embodiment, the invention relates to an immunogenic complex obtained according to the implementation of the method of the invention.

More specifically, it is another object of the present invention to provide an immunogenic complex comprising an immunogen, an antigen or a hapten, wherein said immunogen is conjugated to a support peptide of less than 10 amino acids comprising at least a 3 amino acid residue peptide fragment of the sequence of SEQ ID NO 2.

Preferably, in said immunogenic complex according to the invention, said support peptide comprising at least the peptide encoded by SEQ ID NO2 consists of at most 8 amino acids, preferably at most 5 amino acids, and more preferably 4 amino acids.

According to a preferred embodiment, said support peptide of the immunogenic complex according to the invention consists of the peptide encoded by SEQ ID NO 2.

According to a preferred embodiment, said support peptide of the immunogenic complex according to the invention is characterized in that said binding consists of covalent coupling between said support peptide and said immunogen.

According to a preferred embodiment, the immunogenic complex according to the invention is characterized in that, when the immunogen is a peptide, the support peptide is coupled at the N or C terminus, preferably the N terminus, of the immunogen.

According to a preferred embodiment, the immunogenic complex according to the invention is characterized in that the immunogen is an antigen derived from a bacterium, a parasite and/or a virus.

According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that the immunogen is a surface protein or glycoprotein of Respiratory Syncytial Virus (RSV), in particular F or G, or a sequence having at least 80% identity with the F or G protein sequence, preferably 85%, 90%, 95% or 98% identity with the F or G protein sequence, or a fragment thereof of at least 10 contiguous amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, which upon administration to a mammal is capable of inducing the production of specific antibodies against said fragment.

According to a preferred embodiment, the immunogenic complex according to the invention is characterized in that the immunogen is a human RSV G protein of type a or B or a bovine RSV G protein.

According to a preferred embodiment, said immunogenic complex according to the invention is characterized in that the immunogen is a polypeptide of the sequence between residues 130-230 of the RSV G protein, inclusive of the endpoints, or a polypeptide of the sequence having at least 80% identity with the sequence between said residues 130-230 or a fragment of said sequence between positions 130-230 of the RSV G protein of at least 10 amino acids.

Preferably, the immunogen of the immunogenic complex according to the invention is a polypeptide of the sequence of SEQ id no 3.

According to yet another preferred embodiment, the complex according to the invention is the MEFG2Na complex of the sequence SEQ ID NO4 or a similar immunogenic complex whose sequence has the MEF sequence shown in the sequence SEQ ID NO2 in positions 1 to 3, followed by:

-or a sequence having at least 80% identity to the sequence of SEQ ID NO3, preferably at least 85%, 90%, 95% or 98% identity to the sequence of SEQ ID NO 3;

-or a fragment of the sequence of SEQ ID NO3 of at least 10 contiguous amino acids, preferably at least 15, 20, 25, 30 or 50 amino acids, which is capable of inducing the production of specific antibodies against said fragment upon administration to a mammal.

In a further aspect, it is an object of the present invention to provide a nucleic acid encoding an immunogenic complex according to the invention, in particular an MEFG2Na immunogenic complex encoding the sequence of SEQ ID NO4, said nucleic acid preferably being isolated and/or purified.

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this specification to denote a specific nucleotide sequence, whether modified or not, which defines a segment or region of a nucleic acid, which may or may not contain non-natural nucleotides, and which corresponds to double-stranded DNA, single-stranded DNA or a transcript of said DNA.

In yet another aspect, it is an object of the present invention to provide an immunogenic complex according to the invention or a nucleic acid encoding an immunogenic complex according to the invention, in particular a MEFG2Na immunogenic complex of the sequence of SEQ ID NO4 or a nucleic acid, such as DNA or RNA, encoding said MEFG2Na complex, for use as a medicament.

Pharmaceutical compositions comprising the immunogenic complexes according to the invention or as defined above, or the nucleic acids, RNA or DNA encoding these immunogenic complexes, in association with a physiologically acceptable excipient are also an object of the present invention. The compositions are particularly suitable for the preparation of vaccines.

Immunization may be achieved by administering said polynucleotide encoding an immunogenic complex as defined herein, alone or by a viral vector comprising one such polynucleotide. Host cells, in particular killed bacteria, which have been transformed with one such polynucleotide according to the invention can also be used.

A further object of the invention also includes the use of an immunogenic complex according to the invention, wherein said immunogenic complex is a protein or peptide derived from the RSV G or F protein as defined above, in particular the MEFG2Na complex or one of its analogues 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 RSV-related respiratory infections.

The advantages of the invention will be illustrated by the following examples and the accompanying drawings, in which:

FIG. 1 shows the anti-RSV-AIgG concentrations in mice immunized with BBG2Na or MEFG2 Na;

FIG. 2 also shows in a complementary manner the concentration of anti-RSV-AIgG after a secondary immunization in mice immunized with BBG2Na or MEFG2 Na;

FIG. 3 shows the anti-G2 NaIgG concentration in mice immunized with BBG2Na or MEFG2 Na; and

FIG. 4 also shows in a complementary manner the concentration of anti-G2 NaIgG in mice immunized with BBG2Na or MEFG2 Na.

Example 1: comparison of in vivo Activity induced by BB Carrier protein or MEF supporting peptide

The RSV-ALong strain (10) was administered by nasal route at 20 days⁵pfu) infected 8-week-old IOPS female BALB/c mice. At day 0, after confirmation of RSV-A seroconversion, mice received a single intramuscular injection of 20 μ g BBG2Na adsorbed on Adju-Phos (6 μ g G2Na equivalents) or 6 μ g MEFG2Na adsorbed on Adju-Phos. The concentrations of anti-RSV-AIgG (purified viral antigen) and anti-MEFG 2Na were analyzed by ELISA.

Figures 1 and 2 show that at any point of kinetics there was no significant difference between the concentrations of anti-RSV-AIgG elicited by 6 μ g MEFG2Na or 20 μ g bbg2 Na. The same is true for the concentration of anti-G2 NaIgG (FIGS. 3 and 4).

Example 2: preparation of BBG2Na and MEFG2Na Complex

Preparation of BBG2 Na:

by using Escherichia coli RV308 as host cell and starting with tryptophanThe plasmid controls the transcription of the gene of interest to produce the BBG2Na protein. The fermentation step is a batch process using semi-defined synthetic medium and glycerol as carbon source and energy. For the preparation of the inoculum for the production fermenter, two cultivation steps are necessary. In this fermenter, the microorganism was grown to an optical density of 50 at 620nm and expression was then induced by addition of tryptophan analogue (IAA). Growth continued until O in the fermentor₂The partial pressure suddenly increases, which indicates that the carbon source has been depleted. At this stage, the average cell density was 40g stem cells/liter and there was an expression rate of 9.5%, which represents the productivity of 3.8g BBG2 Na/liter culture. The culture was cooled to +4 ℃ and the microorganisms were collected by centrifugation and cooled at-15 ℃ to-25 ℃.

Extracts of BBG2Na require the dissolution of thawed microbial masses with a buffer containing guanidine, hydrochloric acid and 1, 4-Dithiothreitol (DTT) to reduce disulfide bonds. Renaturation of the protein and oxidation of the disulfide bridges was obtained by diluting the denaturing suspension and shaking overnight at room temperature in an open reactor. The suspension containing renaturated proteins is clarified by centrifugation and subsequent filtration. Next, PEG6000 was added to the filtrate and the resulting precipitate was collected by centrifugation. The BBG2 Na-containing precipitate was redissolved in a urea-containing buffer. The obtained extract is filtered on a 0.22 μm support and stored at-15 deg.C to-25 deg.C.

Purification of BBG2Na from thawed extracts consisted of 5 steps: (1) cation exchange chromatography on a SP-Sepharose Fastflow column; (2) hydrophobic interaction chromatography on a Macro-Prep Methyl column; (3) gel filtration on a Superdex S200 column; (4) anion exchange chromatography on a DEAE-Sepharose Fast Flow column; and finally (5) a desalting step on a Sephadex G25 column. The solution of purified protein was sterile filtered and dispensed into sterile pyrogen-free sachets.

Preparation of MEFG2Na

Production of MEFG2Na protein by Using Escherichia coli icon 200 as host cell and plasmid for controlling transcription of Gene of interest by Tryptophan promoter. Escherichia coli ICONE 200 is a mutant of Escherichia coli RV308 and was developed for improved expression regulation in the growth phase. The fermentation step is a fed-batch (fed-batch) process using chemically defined media and glycerol as carbon source and energy. For the preparation of the inoculum for the production fermenter, two cultivation steps are necessary. In this fermentor, the microorganism was grown to an optical density of 110 at 620am and expression was then induced by addition of tryptophan analog (IAA). Growth continued until O in the fermentor₂The partial pressure suddenly increases, which indicates that the carbon source has been depleted. At this stage, the average cell density was 56g stem cells/liter and had an expression rate of 5.4%, which represents the productivity of 3g MEFG2 Na/liter of culture. The culture was cooled to +4 ℃ and the microorganisms were collected by centrifugation and cooled at-15 ℃ to-25 ℃.

The extract of MEFG2Na requires the lysis of the thawed microbial pellet with a buffer containing guanidine and hydrochloric acid. The suspension containing renaturated proteins is clarified by centrifugation and subsequent filtration. Since guanidine is compatible with subsequent purification steps, buffer exchange is performed using a dialysis concentration step on a polyethersulfone ultrafiltration support with a cut-off threshold of 10 kDa. The resulting extract was filtered on a 0.22 μm support and then purified.

Purification of MEFG2Na consisted of 3 steps: (1) cation exchange chromatography on a Fractogel EMD SE Hicap column; (2) gel filtration on a Superdex 75 Prep Grade column; and (3) anion exchange chromatography on a DEAE-Sepharose Fast Flow column. Bulk purified protein was sterile filtered and dispensed into sterile pyrogen-free sachets.

Expression yield

Data for MEFG2Na and BBG2Na expression are summarized in table 1 below.

Table 1:the amounts of MEFG2Na and BBG2Na protein obtained, expressed in mol/100g of stem cells

mol protein/100 g Stem cells

BBG2Na 2.46×10^-4

MEFG2Na 4.54×10^-4

It showed that the expression rate of MEFG2Na complex was about 2 times higher than that of BBG2Na complex.

Although the present specification, and examples, are based solely on the antigen G2Na, it will be appreciated that any immunogen may be coupled to a support peptide according to the present invention.

Sequence listing

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Claims (20)

1. A method of preparing an immunogenic complex, wherein an immunogen, antigen or hapten is conjugated to a supporting peptide to form said immunogenic complex, wherein said supporting peptide consists of a peptide comprising at least less than 10 amino acids of the peptide represented by the sequence of SEQ id no 2.

2. The method according to claim 1, wherein the support peptide of less than 10 amino acids consists of the peptide encoded by SEQ ID NO 2.

3. The method according to claim 1 or 2, wherein said binding consists of covalent coupling between said support peptide and said immunogen.

4. The method according to claim 3, wherein when the immunogen is a peptide, the support peptide is coupled to the N-terminus of the immunogen.

5. The method according to claim 4, wherein said covalent coupling is carried out by recombinant DNA techniques.

6. The method according to claim 3 or 4, wherein said covalent coupling is carried out by a chemical route.

7. The method according to any one of claims 1 to 6, wherein the immunogen is an antigen derived from a bacterium, a parasite and/or a virus.

8. The method according to claim 7, wherein the immunogen is a surface protein or glycoprotein of Respiratory Syncytial Virus (RSV), a protein having a sequence at least 80% identical to the sequence of said RSV surface protein, or a fragment of at least 10 contiguous amino acids of said RSV surface protein, said protein or said fragment having a sequence at least 80% identical capable of inducing the production of specific antibodies against said protein or said fragment upon administration to a mammal.

9. The method according to claim 8, wherein the immunogen is a human or bovine RSVG protein, type a or B, a protein having a sequence with at least 80% identity to the sequence of said G protein, or a fragment of at least 10 amino acids of said G protein.

10. The method according to claim 9, wherein the immunogen is a polypeptide of the sequence between residues 130-230, including the endpoints thereof, of the RSV G protein or a polypeptide of a sequence having at least 80% identity with the sequence between residues 130-230 or a fragment of at least 10 amino acids of said G protein.

11. The method according to claim 10 wherein the immunogen is a polypeptide of the sequence of SEQ ID NO 3.

12. An immunogenic complex obtained by performing the method according to any one of claims 8 to 11.

13. An immunogenic complex comprising an immunogen, antigen or hapten conjugated to a support peptide, wherein:

-the immunogen is bound, preferably coupled in covalent linkage, to a support peptide of less than 10 amino acids comprising at least the peptide of sequence SEQ ID NO 2; and wherein

-the immunogen is a surface protein or glycoprotein of Respiratory Syncytial Virus (RSV), more particularly F or G, or a sequence having at least 80% identity to the sequence of said RSV surface protein, which upon administration to a mammal is capable of inducing the production of specific antibodies against said protein having a sequence with at least 80% identity.

14. A complex according to claim 13, wherein the support peptide is a peptide of sequence SEQ ID NO 2.

15. Complex according to claim 12 or 13, wherein it is the MEFG2Na complex of the sequence SEQ ID NO4, or an immunogenic complex having the sequence SEQ ID NO2 in positions 1-3, followed by

-a sequence having at least 80% identity to the sequence of SEQ ID NO3, preferably at least 85%, 90%, 95% or 98% identity to the sequence of SEQ ID NO 3.

16. The complex according to claim 15, having the sequence of SEQ ID NO 4.

17. A nucleic acid encoding an immunogenic complex according to any one of claims 13-16.

18. A nucleic acid according to claim 17 encoding an immunogenic complex of the sequence of SEQ ID NO 4.

19. A complex according to any one of claims 12 to 16, or a nucleic acid according to claim 17 or 18, for use as a medicament.

20. Use of an immunogenic complex according to any one of claims 12-16, or a nucleic acid according to claim 17 or 18, for the preparation of a pharmaceutical composition intended for the treatment or prevention of RSV-related respiratory tract infections.