AU8812398A

AU8812398A - Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy

Info

Publication number: AU8812398A
Application number: AU88123/98A
Authority: AU
Inventors: Alain Beck; Liliane Goestch; Thien Ngoc Nguyen; Ultan Power
Original assignee: Pierre Fabre Medicament SA
Current assignee: Pierre Fabre Medicament SA
Priority date: 1997-07-17
Filing date: 1998-07-17
Publication date: 1999-02-10
Anticipated expiration: 2018-07-17
Also published as: CN1264425A; FR2766192B1; WO1999003987A3; EP1003851A2; JP2001510039A; WO1999003987A2; CA2296736A1; AU756110B2; FR2766192A1; BR9810907A

Description

WO 99/03987 - 1 - PCT/FR98/01570 RSV EPITOPES AND ANTIBODIES COMPRISING THEM, WHICH ARE USEFUL IN DIAGNOSIS AND THERAPY The present invention relates to the 5 respiratory syncytial virus, and more particularly to the identification of new epitopes and to the corresponding antibodies, which are useful in particular in the field'of the treatment, prophylaxis and diagnosis of conditions caused by this virus. 10 The respiratory syncytial virus (RSV) is one of the etiological agents most frequently encountered in unweaned babies and in the elderly. The bronchiolites are often serious in children and require hospitalization. Currently, there is no means of 15 prevention against the disease caused by RSV; the first RSV infection does not protect against the next one. The treatment of serious cases with antibiotic therapy (ribavirin) and/or combined with immunotherapy (human immunoglobulins) cannot alleviate the worsening of the 20 disease. Nevertheless, this type of treatment still remains very expensive. Recent clinical trials with the monoclonal antibodies HNK20 from ORAVAX (directed against the RSV F protein) were not effective in the treatment compared with the placebo against the RSV 25 infection in children. During the 60s, attempts to immunize children with an RSV vaccine inactivated with formalin had as a consequence the worsening of the disease instead of conferring protection of the lungs against the natural RSV infection. Associated with this 30 problem, current diagnoses do not make it possible to reliably identify the RSV infection, at least in adults. RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a 35 nonsegmented RNA, of negative polarity, encoding 10 specific proteins. Application WO 87/04185 proposed using RSV structural proteins for a vaccine, such as the envelope proteins called F protein (fusion protein) or G - 2 protein, a glycoprotein of 22 Kd, a protein of 9.5 Kd, or the major capsid protein (N protein). Application WO 89/02935 describes the protective properties of the whole RSV F protein, 5 optionally modified in monomeric or deglycosylated form. A series of fragments of the F protein have been cloned in order to search for their neutralizing properties. 10 In Application WO 95/27787, it has been shown that the G protein of RSV can be used in the preparation of products intended for the treatment and/or prevention of conditions caused by RSV, subgroup A or B. 15 In the context of the present invention, it has now been found that fragments of the G protein of RSV, containing specific epitopes, possess particularly advantageous properties. New peptide fragments of the G protein of RSV can thus be prepared, in particular for 20 the following applications: (i) said peptide fragment coupled or fused, by chemical methods or by genetic engineering, with a carrier, constitutes an effective vaccine against RSV infection, regardless of the mode of administration; 25 (ii) the same peptide fragments can serve to generate polyclonal or monoclonal antibodies which are very effective in the prophylactic or therapeutic treatments of the host infected with RSV; (iii) these peptide fragments and the 30 monoclonal antibodies can be used as reagents in a diagnostic kit making it possible to confirm and to identify the infection in the host infected with RSV-A or RSV-B. The subject of the invention is therefore 35 polyclonal or monoclonal antibodies directed against an epitope of the G protein of RSV corresponding to a sequence chosen from one of the peptide sequences between respectively the amino acid residues 150-159, - 3 176-189, 194-207 and 155-176 of the complete sequence of the G protein of RSV A or B, or of the sequences exhibiting at least 80%, and preferably at least 98% homology. 5 These antibodies will recognize peptides carried by the sequence between amino acid residues 130 and 230 of the G protein of RSV, subgroup A or subgroup B. This region corresponding to amino acids 10 130-230 of the G protein of RSV A is designated hereinafter G2Na. G2Na was produced in a bacterium such as E. coli, and therefore nonglycosylated. It confers, in particular when it is coupled to a carrier such as an OmpA of a gram-negative bacterium, for example 15 Klebsiella (p40 protein, described in WO 96/14415) or a protein derived from Streptococcus (such as the human serum albumin binding protein, called hereinafter BB, described in WO 96/14416), immunological protection against RSV infections. 20 Unexpectedly, it has been shown that a series of peptides prepared according to the invention confers cross-protection against RSV subgroup A or subgroup B. In particular, a recombinant protein BBG2al, a derivative of BBG2Na where only four residues have been 25 modified on G2Na: the residues Asn(aal9l), Lys(aa192), Gly(aa195) and Thr(aa198) have been substituted with Ser, Asn, Lys and Pro residues respectively, confers cross-protection RSV-A or RSV-B in BALB/c mice. With the same aim of enhancing cross-protection, two new 30 molecules were produced: BBG2a2 where the residues Asn(aa157), Asn(aal60), Asn(aal6l) and Phe(aa163) were substituted by the residues Lys, Lys, Asp and Tyr respectively; BBG2a3 comprises the eight modified residues of BBG2al and BBG2a2. 35 Particularly advantageous peptides according to the invention have in particular one of the sequences chosen from the sequences ID No. 1, ID No. 2, ID No. 3, 'sIS ID No. 4, ID No. 5, ID No. 6, ID No. 7, ID No. 8, ID - 4 No. 9, ID No. 10, ID No. 11, ID No. 12, ID No. 13, ID No. 14, ID No. 15, ID No. 16, ID No. 17, ID No. 18, ID No. 19, ID No. 20, ID No. 21 and/or ID No. 22, which are presented in the annex; such a peptide may, in 5 addition, comprise at least one cysteine residue at the N-terminal or C-terminal position. The reactivity of the peptides is demonstrated by mono- or polyclonal probes. Four regions of G2Na were revealed by this technique: G5a(aal44-159), 10 Glla(aal64-176), G4a(aa172-187) and G9a(aal90-204). The subject of the invention is therefore also monoclonal or polyclonal antibodies directed against a peptide having at least one of the sequences ID No. 1 to ID No. 22. 15 Peptides possessing one or more units corresponding to the epitopes 150-159, 176-189, 194-207 and 155-176 of the sequence of the G protein of RSV will be very useful for the different embodiments of the invention. 20 Peptides according to the invention, coupled to a carrier protein, are useful as immunogenic agents. The carrier protein is advantageously chosen from the OmpAs of gram-negative bacteria and fragments thereof, the TT (tetanus toxoid) protein, the human 25 serum albumin binding protein of Streptococcus and fragments thereof and the cholera toxin B (CTB) subunit; preferably, the carrier protein is an OmpA of a bacterium of the genus Klebsiella. According to one of the aspects of the 30 invention, the peptide is conjugated with the carrier protein via a linking protein; this linking protein may in particular be chosen from a mammalian serum albumin receptor and the receptors present at the surface of the mucosal cells. 35 The coupling is preferably a covalent coupling, which may be achieved by the chemical route or by recombinant DNA techniques.

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- 5 According to another of its aspects, the subject of the invention is therefore a nucleotide sequence encoding a peptide or an immunogenic agent as defined above. It may be in particular a hybrid DNA 5 molecule produced by insertion or fusion, in the DNA molecule encoding the carrier protein, of the DNA encoding a peptide according to either of claims 4 and 5 or one of the fragments thereof, fused with a promoter; it may also be an RNA molecule. 10 The peptides, the antibodies, the immunogenic agents and the nucleotide sequences according to the invention may be used as a medicament, and more particularly for the preparation of a composition intended for the preventive or curative treatment of 15 conditions caused by RSV, subgroup A or B. For example, monoclonal antibodies specifically recognizing the G5a and G11a and GlACa peptides have been generated. The passive transfer of the monoclonal antibodies 5C2 (anti-G5a) and 18DI (anti-G1ACa) into 20 naive mice makes it possible to prevent the RSV-A infection on the one hand, and, on the other hand, the same monoclonal 5C2 makes it possible to rapidly eliminate a chronic RSV-A infection in immunosuppressed mice. 25 The subject of the invention is therefore also a pharmaceutical composition, characterized in that it contains at least one mono- or polyclonal antibody, a peptide or an epitope according to the invention, an immunogenic agent, or a nucleotide sequence as defined 30 above, and pharmaceutically acceptable excipients. The monoclonal antibodies are preferably humanized and produced by the recombinant route. According to another aspect of the invention, they are obtained by the phage library method. 35 The peptides, immunogenic agents, antibodies and nucleotide sequences according to the invention can, according to one embodiment of the invention, Ads enter into the composition of _a diagnostic kit. -7-' - 6 As indicated above, the immunogenic agents may be prepared by the recombinant DNA technology, by introducing a nucleotide sequence according to the invention into a host cell. This nucleotide sequence 5 may be a fusion gene which is introduced via a DNA vector which is derived from a plasmid, from a bacteriophage, from a virus and/or from a cosmid. This fusion gene may, in one embodiment of the method of preparation, be integrated into the genome of the host 10 cell. The vector may be a viral vector, known to persons skilled in the art. The host cell may be a prokaryote, in particular chosen from the group comprising: E. coli, 15 Bacillus, Lactobacillus, Staphylococcus and Streptococcus. However, the host cell may also be a yeast, a mammalian cell, a cell of plant origin or an insect cell. 20 According to one of the aspects of the method according to the invention, the fusion protein is expressed: secreted, localized in the cytoplasm or exposed at the membrane of the host cells. The following examples are intended to 25 illustrate the invention. In these examples, reference will be made to the following figures. Figures 1 and 2: Principle of the cloning of the genes G2al, G2a2 and G2a3 into the vectors. Figure 3: A- 20% SDS-PAGE gel, Comassie blue staining. 30 M = molecular size standards, lanes 1 and 2 BBG2al proteins (theoretical mass 38.7 Kd) affinity-purified on HSA-Sepharose. B- Immunoblot of the BBG2al proteins with a monoclonal antibody 18 D1. 35 Figure 4: A- Immunogenicity of the peptides G5a and G9a. B- Protective efficacy of the peptides G5a and G9a on the lungs.

- 7 Figure 5: A- Irmnunogenicity of the peptides G7a and G8a. B- Protective efficacy of the peptides G7a and G8a on the lungs. 5 Figure 6: Immunogenicity of BBG2al with respect to RSV A (Figure 6A) and RSV B (6B) and protective efficacy against RSV A (6C) and RSV B (6D). Figure 7: Curative effect of the monoclonal antibodies 18D1 and 5C2. 10 Figure 8: A- Prophylactic efficacy of the monoclonal antibody 18D1. B- Prophylactic efficacy of the monoclonal antibody 5C2. Figure 9: Identification of the B epitopes of G2Na 15 which are represented in a serum of mice immunized with BBG2Na. A: total staining; B: no staining of the 155-176 region. Figure 10: Determination of the zone of recognition of the monoclonal antibody 5B7 by the Pepscan B method. 20 EXAMPLES Example 1: Synthesis of the peptides * Example of the synthesis of the peptides G5aCys and CysG5a Abbreviations: 25 AA: Amino acid Boc: tert-Butoxycarbonyl BHA: Bromohydrosuccimidyl acid CE: Capillary Electrophoresis ES-MS: Electrospray - Mass spectrometry 30 FMOC: Fluorenylmethoxycarbonyl FZCE: Free Zone Capillary Electrophoresis HBTU: 2- (lH-Bemfo~ztriazole-1-yl) -1, 1,3, 3-tetra methyluronium hexafluorophosphate HMP: p-Hydroxymethylphenoxymethylpolystyrene 35 MBHA: Methylbenzhydrylamine NMP: N-methyl-2-pyrrolidone Pmc: 2,2,5,7, 8-Pentamethylchroman-6-sulfonyl - 8 RP-HPLC: Reverse Phase - High Performance Liquid Chromatography SPPS: Solid Phase Peptide Synthesis tBOC: t-Butyloxycarbonyl 5 tBu: tert-Butyl TFA: Trifluoroacetic acid Trt: Trityl The peptide G5a is a peptide of 16 amino acids which corresponds to the fragment of the G protein 10 (144-159) of RSV-A. It is obtained by solid phase chemical synthesis starting from the C side toward the N-terminal side. The peptides CysG5a and G5aCys correspond respectively to this peptide with an additional Cysteine on the N- or C-terminal side which 15 is intended for a specific coupling with a carrier protein. These two peptides make it possible to study the influence which the orientation of the peptide conjugated with a carrier protein may have on the immunological response observed. 20 The peptides were synthesized with the aid of an automated solid phase peptide synthesizer from the C side toward the N-terminal side (FMOC chemistry at the 0.1, 0.25 or 1.0 mmol scale) . The synthesis of the peptide CysG5a is carried out starting with a Proline 25 preloaded onto a resin of the HMP type, which makes it possible, after cleavage, to obtain a free acid function on the C-terminal side or a resin of the Rink amide MHBA type, which makes it possible, after cleavage, to obtain an amide function on the C-terminal 30 side. That of the peptide G5aCys starts with a Cysteine preloaded onto either of the resins. The reactive functions of the side chains of the amino acids used are protected by groups compatible with the FMOC chemistry [Cys(Trt); Arg(Pmc); Asn(Trt); Gln(Trt); 35 Lys(Boc); Ser(tBu); Thr(tBu)]. A coupling cycle takes place in the following manner: deprotection of the N-terminal amine function of the first amino acid with the aid of piperidine, activation of the acid function - 9 of the second amino acid to be coupled with the aid of HBTU/HMP and coupling. At the end of the synthesis, the peptide is cleaved from the resin and the side chains are deprotected by reacting with a water/TFA mixture. 5 The peptide is precipitated with ether previously cooled to -40 0 C and the mixture is centrifuged. The pellet is washed three times with ether and then dried with nitrogen. The pellet is taken up in water containing 0.1% TFA. The suspension is again 10 centrifuged and the supernatant, which contains the peptide, is separated from the pellet, which contains the resin. The crude peptide is purified by semipreparative reverse phase HPLC. The homogeneity of the purified peptide is checked by reverse phase HPLC 15 and by capillary electrophoresis (FZCE). The theoretical structure is confirmed by comparing the compatibility of the mass measured by ES-MS type mass spectrometry with the mass calculated from the theoretical amino acid sequence. 20 * Peptide CysG5aNH2 Synthesis: Theoretical weight peptide-resin at the end of synthesis: 593 mg Weight measured after 25 drying with nitrogen: 619 mg Cleavage peptide-resin and analysis of the crude peptide: 200 mg (1/3 of the batch) Mass of crude peptide cleaved after 30 lyophilization: 84 mg (75% of net quantity of peptide, that is to say 97% yield) Homogeneity of the crude peptide: 97% (RP-HPLC; UV 210 nm) 97% (FZCE/MicrocoatTM) 35 Purification: Mass of purified peptide after - 10 lyophilization: 50 mg (75% of net quantity of peptide, that is to say 58% yield) Characterization: Homogeneity of the purified 5 peptide: > 99% (RP-HPLC; UV 210 nm) > 99% (FZCE/MicrocoatTM; UV 200 nm) Mass spectrometry (ES-MS) 10 Mass calculated: 1951.29 Da Mass measured: 1950.80 Da ± 0.31 Example 2: Coupling of the peptides G5a, G7a, G8a, G9a, Gla and G11ACa with a carrier protein (P40, BB, TT, 15 KLH) * Example of coupling of the peptide G11ACa with the protein P40 The peptide G11ACa (Seq ID No. 14) is a peptide 20 of 13 amino acids derived from the G protein of RSV. It corresponds to the sequence 164-176 of this protein. During the synthesis, the Cys residue at position 173 was replaced with an Ser residue so as to conserve only one Cys residue at position 176 and to avoid the 25 formation of a 1-2 disulfide bridge which does not exist in the natural G protein (1-4/2-3 pairing). This peptide was coupled with the aid of glutaraldehyde (homobifunctional reagent, coupling with the amine and thiol functions) or in a specific manner 30 with the aid of BHA (heterobifunctional reagent, coupling with the thiol function of the Cysteine at the C-terminal position). : Specific coupling with BHA 35 e Solubilization of the peptide G11ACa 2 mg of G11ACa are solubilized in 2 ml of 0.1M phosphate buffer, pH 7 +0.1% Zwittergent 3-14.

- 11 S Specific coupling with the protein P40 with the aid of a heterobifunctional reagent (BHA) 3 mg of BHA dissolved in 25 pl of DMF are added to 2.5 mg of protein P40, previously dialyzed against a 5 0.1M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14 and stirred for 1 hour at room temperature. After desalting on a PD10 column and diluting with a 0.1M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14, 500-g1 fractions are collected and the tubes containing the 10 bromoacetylated protein are assembled (OD reading at 280 rm) in tubes containing 0.95 ml of solution of the peptide G11ACa are added. The reaction medium is saturated with nitrogen and stirred in the dark for 2 hours at room temperature. The solution is then 15 dialyzed with the aid of a 0.1 M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14 overnight at +4 0 C, with stirring, and the solution obtained is stored frozen. : Coupling with the protein P40 with the aid of a 20 homobifunctional reagent (glutaraldehyde) 10 mg of P40 are dialyzed against a 0.1 M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14. The concentration is adjusted to 2 mg/ml with the aid of a 0.1 M carbonate buffer, pH 9 + 0.1% Zwittergent 3-14. 25 200 mg of SDS of a 4% solution are added. 55.5 pl of glutaraldehyde at 2.5% are added to 2.5 ml of a solution of peptide G11ACa at 1 mg/ml in a 0.1 M carbonate buffer, pH 9 + 0.1% Zwittergent 3-14 at a pH of between 9 and 10. The reaction medium is 30 stirred at +40C for 24 hours and then brought to room temperature. 25 pl of 1 M lysine are added in order to block the reaction. The solution is dialyzed against 0.1 M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14 for 24 hours at + 40C, with stirring. The dialysate is 35 recovered and the SDS removed by precipitation with the aid of a 0.02 M KCl solution 6 times. The last supernatant, which contains the conjugate P40-GlACa glutaraldehyde, is stored in frozen form at -20 0

C.

- 12 * Sterilization of the conjugates The conjugates are thawed, filtered in a sterile manner (0.22 pim), aliquoted and stored at +4 0 C 5 so as to avoid problems of precipitation. * Analytical characterization of the conjugates The conjugates are characterized by assaying the proteins by the Lowry method, SDS-PAGE type 10 electrophoresis (staining with Coomassie blue) and by assaying the amino acids after acid hydrolysis in gaseous phase, derivatization with PITC and analysis by HPLC. Conjugate Volume [prot] No. of G11AC [Gil (ml) mg/ml bound AC] mg/mi rP4O-GllACa 2.7 0.74 2 0.05 (BHA) rP40-GllACa 5.1 1.29 16 0.49 (glutaraldehyde) 15 Example 3: Cloning of the gene G2al, G2a2 into an expression vector pvaBB308 and production of fusion proteins BBG2al and BBG2a2 in E. coli The principle of cloning of the gene G2al 20 (Seq ID No. 15), G2a2 (Seq ID No. 16) and G2a3 (Seq ID No. 17) into the vectors is explained in Figures 1 and 2. 3.1 Construction of G2al 25 The gene encoding the protein G2al (Seq ID No. 15) is constructed by site-directed mutagenesis using, as starting material, the plasmid pRIT28G2Na. For that, two PCR reactions (Polymerase chain reaction) are carried out with the pairs of 30 oligonucleotides RIT29/TH137(PCR 1) on the one hand and - 13 RIT30/TH136(PCR 2) on the other hand under the following conditions: PCR (940C 15 seconds 25 cycles (55 0 C 30 seconds 5 (720C 30 seconds e Attachment onto the magnetic beads The fragments obtained, respectively of 262 bp and 208 bp for reactions 1 and 2, are attached to 10 magnetic beads. 25 pl of DYNAL@ M-280 magnetic beads coupled to streptavidin are rinsed twice beforehand with T.E. buffer (10 mM Tris; 1 mM EDTA, pH 7.5) and then incubated for 20 minutes at 37 0 C with 90 pl of the amplification reactions 1 and 2. After attachment, the 15 fragments are denatured by incubating the magnetic beads with 50 pl of 0.15 M NaOH for 10 minutes at room temperature. The two supernatants are recovered, precipitated with absolute ethanol and resuspended in 50 sl of H 2 0. 20 e Extension reaction This is carried out by PCR by taking 10 pl of each of the amplification reactions under the conditions below: 25 (950C 15 seconds 5 cycles (350C 30 seconds (720C 30 seconds The fragment generated is amplified by PCR with the oligonucleotides RIT27 and RIT28: 30 (95 0 C 15 seconds 25 cycles (550C 30 seconds (720C 30 seconds The amplified fragment of 509 bp is digested with the restriction enzymes PstI and HindIII. The 35 generated fragment of 169 bp is cloned into the vector pRIT28 digested with the same enzymes. The plasmid obtained pRIT28G2al down is sequenced with the Dye Deoxy Terminator chemistry - 14 according to the protocol described by Applied Biosystem (Perkin Elmer). The plasmid pRIT28G2al is obtained by cloning the PstI/HindIII fragment of pRIT28G2aldown (fragment 5 downstream of the Pst I site) into the vector pRIT28G2Na. The G2al gene is then cloned into the expression vector pvaBB308 at the EcoRI/HindIII restriction sites, generating the vector pvaBBG2al. 10 The sequence listing for the oligonucleotides is indicated below: RIT27: 5'-GCTTCCGGCTCGTATGTTGTGTG-3' RIT28: 5'-AAAGGGGGATGTGCTGCAAGGCG-3' 15 TH136: 5'-CCGAAGAAAAAACCGACGACCAAACCGACC-3' TH1 37: 5' -TTTTTTCTTCGGTTTGTTGCTCGGG- 3' RIT29: RIT27 biotinylated in 5' RIT30: RIT28 biotinylated in 5' 20 3.2 Construction of G2a2 (Seq ID No. 16) The principle of the cloning is schematically represented at the bottom of Figure 2. The pairs of oligonucleotides used in this construction are the following: RIT29/TNG193(PCR 1) on 25 the one hand and TNG192/RIT30(PCR 2) on the other hand. The sequences of the oligonucleotides are described below: TNG192: 5' -CCGCCGAAAAAACCGAAAGACGAT-3' TNG193: 5'-CGAAATGGTAATCGTCTTTCGG-3' 30 3.3 Construction of G2a3 (Seq ID No. 17) The two fragments upstream and downstream of the unique PstI site were assembled on the same vector to give pRIT28G2a3. 35 The three fragments of inserts G2al, G2a2 and G2a3 were cloned in various expression vectors into E. coli, in particular in our examples the vectors pvaBB308 where BB is the gene encoding the albumin I< -77 7\S~c - 15 receptor. The fusion proteins obtained BBG2al, BBG2a2 and BBG2a3 can be easily affinity-purified on an HSA Sepharose (Human Serum Albumin) column. 5 3.4 Fermentation and purification of fusion proteins BBG2al and BBG2a2 E. coli RV308 transformed with the plasmids pvaBBG2al and pvaBBG2a2 are inoculated, respectively, into two Erlenmeyer flasks containing 250 ml of TSB 10 (Tryptic Soy Broth, Difco) medium and Ampicillin (100 pg/ml, Sigma) and tetracycline (8 pg/ml, Sigma) . The medium is incubated for 16 hours at TO = 37 0 C, with stirring. 200 ml of this culture are inoculated into a fermenter (CHEMAP CF3000, ALFA LAVAL) containing 15 2 liters of culture medium. The medium contains (g/1) = glycerol 5; ammonium sulfate, 2.6; potassium dihydrogen phosphate, 3; dipotassium hydrogen phosphate, 2; sodium citrate 0.5; yeast extract, 1; ampicillin, 0.1; tetracycline 0.008; thiamin, 0.07; magnesium sulfate, 1 20 and 1 ml/l of solution of trace elements and 0.65 ml/l of solution of vitamins. The parameters controlled during the fermentation are: the pH, the stirring, the temperature, the rate of oxygenation, the supply of combined sources (glycerol or glucose) . The pH is 25 regulated at 7.0. The temperature is set at 37 0 C. The growth is controlled by supplying glycerol (87%) at a constant flow rate (12 ml/h) in order to maintain the dissolved oxygen pressure signal at 30%. When the turbidity of the culture (measured at 580 nm) reaches 30 the value of 80 (after about 24 hours of culture), the production of proteins is induced by addition of indoleacrylic acid (IAA) at the final concentration of 25 mg/l. About 4 hours after induction, the cells are harvested by centrifugation. The biomass yields 35 obtained are about 200 g of wet biomass. The yields of production of BBG2al and BBG2a2 are about 4 to 6 mg of proteins per g of biomass. - Th.

- 16 A fraction of 30 g of wet biomass is resuspended in 70 ml of TST solution (Tris-HCl 50 mM pH 8.0, 200 mM NaCl, 0.~05% Tween 20 and 0.5 mM EDTA) . The cells are disintegrated by sonication (Vibracell 5 72401, Sonics & Materials). After centrifugation of the cell lysate, the supernatant is filtered (1.2 Jim) and diluted in 500 ml of TST. The fusion proteins thus obtained in soluble form are purified on an affinity column: HSA-Sepharose (human serum albumin) according 10 to the protocol described by (Stahl et al., J. Immunol. Methods, 1989; 124: 43-52). The insoluble lysate, after centrifugation, is washed once with a buffer (50 mM Tris-HCl, pH 8.5; 5 mM MgCl 2 ). After washing, the pellet is solubilized in 15 30 ml of 7 M guanidine hydrochloride, 25 mM Tris-HCl (pH 8.5), 10 mM dithiotreitol (DTT), followed by incubation at 370C for 2 hours. The solubilized proteins are added to a renaturation buffer (25 mM Tris-HCl (pH 8.5); 150 mM NaCl and 0.05% Tween 20). The 20 concentration of guanidine hydrochloride is adjusted to the final concentration of 0.5 M in the renaturation buffer before the addition of the solubilized fusion proteins. The mixture is incubated at room temperature, with moderate stirring, for 16 hours. After 25 centrifugation, the fusion products soluble in the supernatant are purified on an HSA-Sepharose column. The purified fusion proteins are analyzed on an SDS-PAGE gel (12%), on the MINI PROTEAN II SYSTEM apparatus (BIORADS) . The proteins are visualized with 30 Coomassie brilliant blue R250. Furthermore, analysis of the recombinant proteins by immunoblotting with antibodies specific for RSV shows that the proteins are antigenic (see example of SDS gel and immunoblot of BBG2al in Figure 3).

- 17 Example 4: Immunogenecity and protective efficacy of the peptides G5a and G9a coupled to P40 Materials and methods Groups of 7 mice were immunized twice by the 5 i.p. route with 20 sg of P40-G9aCys, P40-CysG5a, or P40-G5aCys. Control mice were immunized with PBS. Alhygrogel (20% v/v) was used as adjuvant for all the immunizations. Samples were taken from the mice at the retroorbital sinus 2 weeks after the last immunization 10 in order to confirm their seroconversion with respect to RSV-A, they were challenged one week later with 105

TCID

50 RSV-A by the i.n. route, and sacrificed 5 days post-challenge. The lungs were removed and the virus titer in the lungs determined. 15 Results Humoral immune responses As indicated in Fig. 4A, the immunogenicity of the peptide G5a is dependent on the orientation of the coupling to P40. After coupling by the C-terminal part 20 of the peptide, low to moderate anti-RSV-A antibody titers were induced in the serum. On the other hand, after coupling by the N-terminal part, G5a did not induce such antibodies. In general, the peptide G9a, coupled at the C-terminal part to P40, was weakly 25 immunogenic in terms of induction of anti-RSV-A antibodies. One mouse out of seven which were immunized with P40-G9acys developed a high anti-RSV-A serum antibody titer. 30 Protective efficacy of the peptides G5a and G9a on the lungs As demonstrated in Fig. 4B, and in direct correlation with the detection of the anti-RSV-A antibodies, the peptide G5a induced protective immune 35 responses when it was coupled at the C-terminal part, but not after coupling at the N-terminal part. Indeed, 6 mice out of 7 immunized with P40-G5acys (C-terminal coupling) were protected without evidence of virus in - 18 the lungs, the last one only had the virus at the assay detection limit. On the other hand, the mice immunized with P40-cysG5a (N-terminal coupling) had virus titers in the lungs which were as high as the control mice 5 immunized with PBS. These results confirm that the orientation of the coupling of this peptide to a carrier protein is essential for its protective efficacy. The correlation between protection and induction of the anti-RSV-A antibodies suggests that 10 the pulmonary protection observed is mediated, at least in part, by the antibodies. Despite the fact that P40-G9aCys was weakly immunogenic in mice in terms of induction of anti-RSV-A serum antibodies, the lungs of 5 mice out of 7 were 15 protected against a challenge with RSV-A. Indeed, 1 mouse out of 7 showed no evidence of virus in the lungs, or even of anti-RSV-A antibodies in the serum. Conclusions 20 The peptides G5a and G9a contain protective epitopes against an RSV-A infection in the lungs. The orientation of the coupling of G5a to a carrier protein is essential for its protective efficacy. 25 Example 5: mmunogenicity and protective efficacy of the peptides G7a and G8a. Materials and methods Groups of 3 to 4 mice were immunized twice by the i.p. route with 20 pg of G7a, G8a, BB-G7a, or 30 BB-G8a. Control mice were immunized with PBS. Alhygrogel (20% v/v) was used as adjuvant for all the immunizations. Samples were taken from the mice at the retroorbital sinus 2 weeks after the last immunization in order to confirm their seroconversion with respect 35 to RSV-A, they were challenged one week later with 105

TCID

5 0 RSV-A by the i.n. route, and sacrificed 5 days post-challenge. The lungs were removed and the nasal - 19 tracts washed. The virus titers in the lungs and the nasal tracts were determined. Results Humoral immune response. 5 As indicated in Fig. 5A, the peptides G7a and G8a are both immunogenic with respect to RSV-A, coupled or otherwise to BB. If the serum antibody titers are considered, the noncoupled peptides appear to be slightly more immunogenic than the coupled peptides. 10 Protective efficacy of the peptides G7a and G8a on the lungs. As indicated in Fig. 5B, the lungs of all the mice immunized with the peptides G7a or G8a, coupled or otherwise to BB, are protected against a challenge with 15 RSV-A, with no presence of virus or only at the assay detection limit. x7 T-

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- 20 Conclusions. The peptides G7a and G8a contain epitopes which are protective with respect to the lungs. The peptides are effective coupled or otherwise to BB. 5 Example 6: Immunogenicity and protective efficacy of the fusion protein BBG2a1. Materials and methods. In order to determine the immunogenicity and 10 protective efficacy of BBG2al against RSV-A and B, groups of 3 mice were immunized twice and three times, respectively, by the i.p route with 20 pg of protein at an interval of 2 weeks. Control mice were immunized with PBS. Alhygrogel (20% v/v) was used as adjuvant for 15 all the immunizations. Samples were taken from the mice at the retroorbital sinus 2 weeks after the last immunization in order to confirm their seroconversion with respect to RSV-A, they were challenged one week later with 105 TCID 50 RSV-A by the i.n. route, and 20 sacrificed 5 days post-challenge. The lungs were removed and the virus titer in the lungs determined. Results. Immunogenicity of BBG2al with respect to the RSV-A and B._ 25 The results presented in Figs. 6A and B demonstrate that BBG2al is capable of inducing anti RSV-A and B antibodies. As expected, the anti-RSV-A antibody titer is much higher than the anti-RSV-B antibody titer. Nevertheless, titers of antibodies 30 against the two strains of RSV are high. Protective efficacy of BBG2al with respect to RSV-A and RSV-B. As indicated in Figs. 6C and D, the immunization of the mice with BBG2al induced immune 35 responses capable of protecting the lungs against a challenge with RSV-A and against a challenge with RSV-B. The mice challenged with RSV-A were protected without evidence of virus in the lungs (2 mice/3) or - 21 only at the detection limit (1 mouse/3). The mice challenged with RSV-B were protected, either without evidence of virus in the lungs (1 mouse/3) or with virus only at the detection limit (1 mouse/3) or just 5 above this detection limit (1 mouse/3). Conclusions. The fusion protein BBG2al is highly immunogenic with respect to RSV-A and with respect to RSV-B. BBG2al 10 is capable of inducing responses which protect the lungs against a challenge with the 2 subgroups of RSV. Example 7: Production of the antibodies 18D1, 5C2 and 5B7 15 Immunization peptide: (i) GlACa coupled to KLH (Keyhole Lempet Haemocyanin), (ii) G2ACa and (iii) BBG2Na. The mice were immunized at DO with 50 pg of antigen in CFA (complete Freund Adjuvant) by the ip 20 route, at D14 with 10 gg of each antigen in IFA (Incomplete Freund Adjuvant) by the ip route, and then at D38 by the iv route with 10 pg of each peptide without adjuvant. The spleens are removed and fused with the myeloma cells SP2-0 at D42 in a 1/1 ratio. The 25 hybridomas which are positive against each antigen are kept. These hybridomas were injected into mice in order to obtain ascites and then the different antibodies obtained were selected on different peptides in order to determine the specificity of the antibodies 30 obtained. The monoclonal antibodies 18D1 and 5C2 selected by their specificity against the peptides GlACa and G5a specifically recognize RSV-A. The monoclonal antibody 5B7 obtained from BBG2Na and recognizing the peptide Glla recognizes RSV-A.

- 22 Example 8: Curative effect of the monoclonal antibodies 18D1 and 5C2 on chronic RVS-A infection obtained in SCID mice. Materials and methods. 5 The C.B-17 scid/scid mice were challenged by the i.n. route with 105 TCID 50 of RSV-A, in 50 pl. Twenty-six days later, the mice received by the i.n. route and at the rate of 7 mice per group 50 pl of a preparation of antibody 18D1 or of antibody 5C2 at an 10 anti-RSV-A ELISA titer of 10 4 . Control mice received anti-BB serum at an anti-BB ELISA titer of 104. The mice were sacrificed 5 days later and their lungs were removed for titration of the virus. 15 Results. The results presented in Fig. 7 demonstrate that the monoclonal antibodies 18D1 and 5C2 are capable of eliminating a chronic infection with RSV-A in SCID mice, in a sterilizing manner. No trace of virus was 20 detected in the lungs at the time of sacrificing. The results obtained in the lungs of mice treated with 5C2 correspond to the mean of the assay detection limits, a limit which is higher because of the lack of availability of sample, and not the presence of virus. 25 Conclusions. The monoclonal antibodies 5C2 and 18D1 could be used as therapeutic treatment in the context of pulmonary RSV-A infections. 30 Example 9: Prophylactic effect of the monoclonal antibody 18D1 on the RSV-A infection in mice. Materials and methods. A group of naive BALB/c mice, which are sero negative with respect to RSV-A, received by intra 35 peritoneal injection 200 pl of a preparation of antibody 18D1 adjusted to the anti-RSV-A ELISA titer of 105. A group of i.p. transfer control mice received in parallel 200 gl of a preparation of anti-P40 serum S T/> - 23 (irrelevant serum) adjusted to the anti-P40 ELISA titer of 10 4 . All the mice are infected the next day by the i.n. route with 50 pl 6f a viral suspension containing 105 TCID 5 o of RSV-A. They are sacrificed 5 days later to 5 assay the virus in the lungs. Results. As indicated in Fig. 8A, the antibody 18D1 is capable, after i.p. transfer, of inducing protection at 10 the level of the lungs of naive mice during a challenge with RSV-A. All the mice are protected after injection of 200 pl of 18D1 at the titer of 10 5 . Three mice out of 7 are protected without evidence of virus in the lungs. The others show traces of virus only at the assay 15 detection limit (3 mice/7), or just above this limit (1 mouse/7). The control mice have titers of between logio 3.70 and 4.45 per gram of lungs. Conclusion. 20 The antibody 18D1 is capable of preventing a pulmonary RSV-A infection in the BALB/c mouse. It demonstrates a substantial prophylactic efficacy. Example 10: Prophylactic effect of monoclonal antibody 25 5C2 on the RSV-A infection in mice. Materials and methods. Naive mice which are seronegative with respect to RSV-A receive by i.n. transfer 50 pl of a preparation of 5C2 adjusted to the anti-RSV-A ELISA 30 titer of 104. Control mice receive in parallel anti-BB serum adjusted to the anti-BB ELISA titer of 104. All the mice are infected the next day by the i.n. route with 50 l of a viral suspension containing 101 TCID 5 0 of RSV-A. They are sacrificed 5 days later 35 for titration of virus in the lungs. Results. As shown in Fig. 8B, all the mice treated with 5C2 at 104 were protected at the pulmonary level. Only 1 S 1<4 - 24 mouse out of 7 shows traces of virus. The control mice have titers of between logio 3.70 and 4.70 per gram of lungs. Conclusion. 5 The antibody 5C2 is capable of preventing a pulmonary RSV-A infection in BALB/c mice. It demonstrates a substantial prophylactic efficacy. Example 11: Pepscan: Example of the multiple synthesis 10 of 94 octapeptides covering the sequence of the aa 130-230 of G2Na and overlapping by one amino acid. A "PEPSCAN" plate of 96 octapeptides synthesized in parallel (2 control peptides + 94 peptides covering the sequence 130-230 of the G protein 15 of RSV-A) is prepared according to the MULTIPINTM technique described by Geysen et al., Proc. Natl. Acad. Sci., 1984, 81, 3998-4002. These peptides are synthesized on a solid support at the end of 96 complementary "pins" (8 x 12) 20 of an ELISA microtiter plate in which the screening of monoclonal or polyclonal antibodies (sera) will be directly carried out. S System for identifying the positions of the pins and 25 of the wells The numbering system used is the following: position A1(1) = plate No. A, line 1 (12 lines) column 1 (position Hi in ELISA) position A2(1) = plate No. A, line 2, column 1 30 (position H2 in ELISA) Al(1): control peptide # 1: PLAQGGGG A2(1): control peptide # 2: GLAQGGGG A3(l): octapeptide # 1: TVKTKNTT A4(l): octapeptide # 2: VKTKNTTT 35 etc. A12(8): octapeptide #94: KEVPTTKP - 25 Synthesis The synthesis corresponds to several cycles of deprotection, washes and coupling until the desired peptide sequences are obtained. At the end of the 5 synthesis, the peptides are N-acetylated, before the step of deprotecting the side chains. The amino acids used for the synthesis on pins are protected by an Fmoc (9-Fluorenylmethoxycarbonyl) group and the following protecting groups for the side 10 chains: t-Butyl ether (tBu) for Serine, Threonine and Tyrosine, t-Butyl ester (OtBu) for Aspartic and Glutamic Acids, t-Butoxycarbonyl (Boc) for Lysine, Histidine and Tryptophan, 2,2, 5,7, 8-pentamethylchroman 6-sulfonyl (Pmc) for Arginine, Trityl (Trt) for 15 Cysteine, Asparagine and Glutamine. The activation of the acid function is carried out with diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) dissolved in DMF. 20 o Fmoc-deprotection and washings: The plate of pins is immersed in a bath containing 200 ml of a solution of piperidine at 20% in DMF (40/160 ml) for 20 min at room temperature, with stirring. The pins are then removed from the bath. The excess solvent is removed by hitting 25 the block of pins on paper cloth. The pins are washed in 200 ml of DMF for 2 min at room temperature, with stirring. The block is hit on paper cloth and immersed completely in an MeOH bath for 2 min, without stirring. The pins are then immersed in 200 ml of MeOH (3 baths 30 of 2 minutes, 200 ml/bath). The plate is dried for 30 min. e Coupling of the Fmoc-amino acids: The Fmoc-amino acids undergo a step of activation before they can be 35 coupled. The duration of a coupling step is 2 hours for a concentration of 100 mM with 1.5 equivalents of HOBt and 1.2 equivalents of DIC. A software makes it possible to calculate the _ quantities of reagents - 26 necessary for the coupling step. The weighings are carried out in tubes arranged in alphabetical order of the one-letter code for amino acids. The tubes are filled off the scale on a sheet of paper cloth and then 5 weighed until a mass close to that indicated on the weighing sheet is obtained. The mass should not be 0.2 mg lower or 0.9 mg higher relative to the theoretical quantity. 10 e Coupling step: The pins are gently introduced into the wells. The box is carefully closed and left in a fume cupboard for the duration of the coupling, for 2 h for an amino acid concentration of 100 mM. A 2-h coupling makes it possible to carry out 3 couplings per 15 day. o Treatment of the blocks of pins after coupling: The pins are removed from the plate containing the coupling solutions and washed with 200 ml of MeOH, with 20 stirring, for 5 min. The block is hit on paper cloth and left to dry for 2 min. The block is placed in 200 ml of DMF and washed for 5 min, with stirring, before carrying out the next cycle of deprotection. The plate is conventionally washed and undergoes the step 25 of cleavage of the Fmoc groups as described above after the last coupling. e Acetylation of the terminal amines: The pin heads are incubated in the wells of a plate containing 150 pl of 30 the following reactive mixture: DMF/acetic anhydride/triethylamine 50/5/1 (v/v/v). The block is enclosed in a box for 90 min at room temperature. The block is then washed with 200 ml of MeOH for 15 min and then dried for 15 min. 35 e Deprotection of the side chains: The groups protecting the side chains are removed by immersing the pins in 200 ml of a TFA/anisole/ethanedithiol - 27 190/5/5 ml mixture for 2 h 30 min, at room temperature. After this deprotection step, the block of pins is removed from the acid solution. The box is rinsed once with MeOH, and then filled with MeOH so that the block 5 of pins is completely immersed therein for 10 min. The block is then hit on paper cloth and then immersed in 200 ml of an MeOH/water/acetic acid (100/100/1 ml) mixture for 1 hour and again hit on paper cloth. The block is placed under vacuum in a desiccator overnight. 10 Example 12: ELISA The plate carrying the pins is saturated for 1 hour at 37 0 C in saturation buffer (PBS, 0.1% Tween, 1% gelatin), washed for 10 min in PBS and incubated 15 overnight at 40C, with stirring, with the serum to be analyzed, diluted beforehand. The plate is then washed 4 times 10 min in PBS and incubated for one hour at room temperature with a secondary antibody labeled with peroxidase, diluted 1/5000. After 4 washes in PBS, the 20 TMB substrate is added. The reaction is stopped by addition of sulfuric acid. The summary of the data resulting from the analysis of an anti-BBG2Na murine serum by the Pepscan B method is represented in Figure 9. 25 Figures 9A and 9B show an anti-BBG2Na mouse serum reactivity against 4 regions of the G2Na molecule (the residues in the bold line represent the amino acids which play an important role in the Ac/Ag recognition): 30 - region 1 situated between residues 150 and 159 whose sequence is QRQNKPPNKP. This region is contained in the peptide G5a (144-159) and corresponds to the zone of reactivity of the monoclonal antibody 5C2; - region 2 situated between residues 176 and 189 35 whose sequence is CSNNPTCWAICKRI. It is a region located at the level of the peptide GlACa (174-187) and corresponding to the reactivity of the monoclonal antibodies 18D1 and 5D3; - 28 - region 3 situated between residues 194 and 207 whose sequence is PGKKTTTKPTKKPT. This sequence corresponds to a reactivity at the level of the peptide G9 (194-204), a reactivity already demonstrated during 5 the production of monoclonal antibodies directed against BBG2Na; - region 4 which extends over a wide zone going from residue 155 to residue 176 appears to be the result of different reactivities. One of these reactivities which 10 covers a very hydrophobic region of the molecule G2Na (Glla) corresponds to the zone of recognition of the monoclonal antibody 5B7 (obtained after immunization of BALB/c mice with the candidate vaccine BBG2Na) whose pepscan B is represented below in Figure 10. 15 This monoclonal antibody recognizes the sequence FEVFNFVP (165-172). All the four reactivities above have moreover been confirmed by titration of the anti-BBG2Na serum by means of different ELISA developed for each of the 20 reactivities. Table I shows that the anti-BBG2Na serum indeed exhibits "anti-G4a, . G5a cys, G9a cys and Glla" activities. Table I: anti-G4a, G5aCys, G9aCys and G11ACa titers 25 expressed as logo of the anti-BBG2Na serum ref. BE-02. Titer (logio) BE - 02 G2Na 5.9 KLH-G4a 4.7 KLH-G9aCys 5.0 KLH-G5Acys 3.8 P40-G11ACa 3.5 Conclusion: The study of an anti-BBG2Na serum by the 30 Pepscan technique shows that-the immunization of mice - 29 with BBG2Na generates antibodies against 4 B epitopes situated respectively in regions 150-159, 176-189, 194-207 and 155-176. This technique therefore makes it possible to confirm the importance of region 164-176 5 (G11ACa). These results are moreover in perfect agreement with the ELISA data relating to the reactivity of an anti-BBG2Na serum on the peptides G4a, G5aCys, G9aCys and G11ACa. S >T

Claims

1. Polyclonal or monoclonal antibodies directed against an epitope of the G protein of RSV corresponding to a sequence chosen from one of the peptide sequences between respectively the amino acid residues 150-159, 176-189, 194-207 and-1\55-176 of the complete sequence of the~G protein of RSV A or B, or of the sequences exhibiting at least 80% homology.

2. Antibodies according to claim 1, characterized in that they are directed against a peptide carried by the sequence between amino acid residues 130 and 230 of the G protein of RSV, subgroup A or subgroup B.

3. Antibody according to either of claims 1 and 2, characterized in that it is directed against a peptide having at least one of the sequences ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5, ID No. 6, ID No. 7, ID No. 8, ID No. 9, ID No. 10, ID No. 11, ID No. 12, ID No. 13, ID No. 14, ID No. 15, ID No. 16, ID No. 17, ID No. 18, ID No. 19, ID No. 20, ID No. 21 and/or ID No. 22.

4. Peptide capable of generating an antibody according to one of claims 1 to 3, characterized in that it has at least one of the sequences chosen from the sequences ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5, ID No. 6, ID No. 7, ID No. 8, ID No. 91 ID No. 10, ID No. 11, ID No. 12, ID No. 13, ID No. 14, ID No. 15, ID No. 16, ID No. 17, ID No. 18, ID No. 19, ID No. 20, ID No. 21 and/or ID No. 22.

5. Peptide according to claim 4, characterized in that it comprises, in addition, at least one cysteine residue at the N-terminal or C-terminal position.

6. Immunogenic agent, characterized in that it comprises at least one peptide according to either of claims 4 and 5, coupled to a carrier protein.

7. Immunogenic agent according to claim 6, characterized in that the carrier protein is chosen from the OmpAs of gram-negative bacteria and fragments REPLACEMENT SHEET (RULE 26) - 31 thereof, the TT protein, the human serum albumin binding protein of Streptococcus and fragments thereof and the cholera toxin B(CTB) subunit.

8. Immunogenic agent according to either of claims 5 6 and 7, characterized in that the carrier protein is an OmpA of a bacterium of the genus Klebsiella.

9. Immunogenic agent according to one of claims 6 to 8, characterized in that the peptide is conjugated with the carrier protein via a linking protein.

10 10. Immunogenic agent according to claim 9, characterized in that the linking protein is chosen from a mammalian serum albumin receptor and the receptors present at the surface of the mucosal cells.

11. Agent according to one of claims 6 to 10, 15 characterized in that the coupling is a covalent coupliin .

12. Nucleotide sequence encoding an immunogenic agent according to one of claims 6 to 11.

13. Nucleotide sequence according to claim 12, 20 characterized in that it is a hybrid DNA molecule produced by insertion or fusion, in the DNA molecule encoding the carrier protein, of the DNA encoding a peptide according to either of claims 4 and 5 or one of the fragments thereof, fused with a promoter. 25

14. Nucleotide sequence according to claim 12, characterized in that it is an RNA molecule.

15. As medicament, antibodies according to one of claims 1 to 3, peptide according to either of claims 4 and 5, immunogenic agent according to one of claims 6 30 to 11, or nucleotide sequence according to one of claims 12 to 14.

16. Pharmaceutical composition, characterized in that it contains at least one antibody according to one of claims 1 to 3, a peptide according to either of 35 claims 4 and 5, an immunogenic agent according to one of claims 6 to 11, or a nucleotide sequence according REPLACEMENT SHEET (RULE 26) - 32 to one of claims 12 to 14, and pharmaceutically acceptable excipients.

17. Use of at least one antibody according to one of claims 1 to 3, of at least one peptide according to 5 one of claims 4 and 5, immunogenic agent according to one of claims 6 to 11, or nucleotide sequence according to one of claims 12 to 14, for the "preparation of a composition intended for the preventive or curative treatment of conditions caused by RSV, subgroup A or B. 10

18. Diagnostic kit, characterized in that it comprises an antibody according to one of claims 1 to 3, a peptide according to either of claims 4 and 5, an agent according to one of claims 6 to 11 or a nucleotide sequence according to one of claims 12 to 15 14.

19. Method of preparing an immunogenic agent according to one of claims 6 to 11, characterized in that the coupling between the peptide and the carrier protein is achieved by the chemical route.

20 20. Method of preparing an immunogenic agent according to one of claims 6 to 11, characterized in that the coupling is achieved by the recombinant DNA technology.

21. Method of preparation according to claim 19, 25 characterized in that it comprises a step during which a nucleotide sequence according to one of claims 12 to 14 is introduced into a host cell.

22. Method according to claim 21, characterized in that the nucleotide sequence is a fusion gene which is 30 introduced via a DNA vector which is derived from a plasmid, from a bacteriophage, from a virus and/or from a cosmid.

23. Method according to either of claims 21 and 22, characterized in that the fusion gene is integrated 35 into the genome of the host cell.

24. Method according to one of claims 21 to 23, characterized in that the host cell is a prokaryote. REPLACEMENT SHEET (RULE 26) - 33

25. Method according to claim 24, characterized in that the host cell is chosen from the group comprising: E. coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus. 5

26. Method according to claims 21 to 23, characterized in that the host cell is a yeast.

27. Method according to claims 21 to 23, characterized in that the host cell is a mammalian cell. 10

28. Method according to claims 21 to 23, characterized in that the host cell is a cell of plant origin.

29. Method according to claims 21 to 23, characterized in that the host cell is an insect cell. 15

30. Method according to claims 21 to 23, characterized in that a viral vector is used.

31. Method according to one of claims 20 to 27, characterized in that the fusion molecule is expressed, anchored and exposed at the membrane of the host cells. 20

32. Composition useful according to claim 16 in that the monoclonal antibodies are humanized and produced by the recombinant route.

33. Composition useful according to claim 16 in that the monoclonal antibodies are obtained by the 25 phage library method. REPLACEMENT SHEET (RULE 26)