AU764061B2 - Use of active P40 conjugates for nasal delivery - Google Patents

Description

WO 99/49892 PCT/FR99/00703 USE OF ACTIVE P40 CONJUGATES FOR NASAL DELIVERY The present invention relates to the production of immunizing preparations which are effective in nasal administration. It thus relates to the use of carrier proteins which can improve the immune response to a hapten when the hapten/carrier protein conjugate is administered nasally.

The use of vaccine for oral or nasal delivery is thought to have a great influence on the eradication of pathogenic microorganisms. Specifically, any modification of a vaccine which allows it to be used with greater flexibility (heat-stability, distribution without syringes, etc.), would result in a more effective and more widely used vaccination. On the other hand, immunization via the mucous membrane pathways makes it possible to induce a local immunity which constitutes the first barrier against invasion by a microorganism.

Currently, the oral vaccines which are on the market only concern attenuated or recombined live vectors: tetravalent oral vaccine against polio, oral vaccine against typhoid fever.

Approaches for nasal or oral vaccination are already described in the literature.

Tests have thus been carried out on mucosal administrations of PspA, which corresponds to the surface protein A of Pneumococcus (Briles patent EP 0,682,950), on hemaglutinin filaments (Capron A., patent FR 2,718,750; Kimura patent EP 0,471,177; Shahin US patent 7532327), on a fragment of the tetanus toxin (Dougan patent WO 93/21950) and on cholera toxin B (CTB).

A protein of the external membrane of Neisseria meningitidis is used, mixed with the hapten as an adjuvant for a nasal immunization (Van de Verg L.L., Infection and immunity, 1996, 64: 5263-5268).

05-JUN-2003 18:15 P.10/14 wo 99/49892 2 PCT/FR99/00703 Unexpectedly, the Applicant has now found that a membrane protein originating from another bacterium makes it possible, when it is administered nasally together with an antigen, to induce an immune response of satisfactory strength and quality for the production of a vaccine.

For this reason, the subject' of the present invention is the use of at least one fragment of an enterobacterium membrane protein OmpA for preparing a pharmaceutical composition adaptedfor nasal administration to improve the immunity of a mammal with respect to an antigen or to a hapten.

In the present invention, the term "OmpA" is intended to refer to the type A proteins of the external membrane (OmpA for Outer membrane protein A).

A subject of the invention is also the use of at, least one fragment of a membrane protein of Klebsiella for preparing a pharmaceutical composition intended to be administered nasally, to improve the 20 immunity of a mammal with respect to an antigen or to a r hapten.

Preferably, the membrane protein is an OmpA protein of Klebsiella pneumoniae.

Advantageously, said fragment of the enterobacterium membrane protein OmpA or of the Klebsiella membrane protein according to the invention is obtained by recombinant process.

Very advantageously, said membrane protein or its fragment, obtained by recombinant process, is, 30 after extraction, renatured in the presence of detergent chosen from Zwittergent 3-14, Zwittergent 3-12 and octylglycopyranoside, preferably in the oe presence of Zwittergent 3-14 at a concentration of between 0.05% and 2% very preferably at a 35 concentration close to 0.1%.

Application WO 96/14415 has shown that the major membrane protein of Klebsiella pneumoniae, which is the OmpA named P40, coupled to peptide subunit antigens is very immunogenic via the systemic route.

COMS ID No: SMBI-00283893 Received by IP Australia: Time 18:15 Date 2003-06-05 WO 99/49892 3 PCT/FR99/00703 The recombinant P40 protein, expressed in E.Coli in the form of inclusion bodies, is named In the context of the present invention, a particularly suitable protein comprises the sequence SEQ ID No 1.

The Applicant has demonstrated that an antibody response is found in all adults, the enterobacterium Klebsiella pneumoniae being a very widespread pathogen. This sensitization favors an increase in the antibody response directed against an antigen or a hapten which is administered while coupled to the carrier protein P40. The administration is carried out nasally in the absence of adjuvant.

Said antigen or hapten according to the invention can be chosen from the group comprising proteins, peptides, polysaccharides, oligosaccharides and nucleic acids. Advantageously, it is of bacterial or viral origin.

The present invention is thus suitable for preparing vaccine directed against any microorganism responsible for pathologies of the airways, such as for example microorganisms chosen from RSV, parainfluenzae virus (PIV), influenza virus, hantavirus, streptococci, pneumococci and meningococci.

The antigen or hapten according to the invention will comprise at least one fragment of said microorganism, such as a protein fragment, which persons skilled in the art will know how to determine for its capacity to confer the desired immunity, by standard techniques such as those described in the examples below.

In particular, the present invention is suitable for preparing vaccine directed against RSV (or respiratory syncytial virus), in particular human or bovine RSV. In this case, the antigen or hapten according to the invention comprises at least one protein fragment of the virus RSV, and in particular at least one fragment of the protein G of the RSV.

WO 99/49892 4 PCT/FR99/00703 The sequences of such fragments have in particular been described in application WO 95/27787.

Preferably, said protein fragments of the virus RSV are chosen from the fragments having the sequences SEQ ID No 2 to SEQ ID No 74 as amino acid sequences.

Sequences which are suitable for preparing a vaccine according to the invention are the sequences SEQ ID No 2 to SEQ ID No 74.

The chemical conjugates derived from the coupling of peptides to at least one fragment of a membrane protein of Klebsiella, such as rP40, give good results, and an evaluation of the immune response shows very strong antibody responses against these peptides after presensitization with Klebsiella pneumoniae.

Advantageously, the protein fragment originating from enterobacteria membrane protein OmpA or from membrane protein of Klebsiella is covalently coupled to the antigen or hapten, such as a protein fragment of the RSV.

The invention also comprises the use of at least one fragment of an enterobacteria membrane protein OmpA or of a membrane protein of Klebsiella according to the invention, characterized in that said fragment is covalently coupled to said antigen or hapten.

According to the invention, it is possible to introduce one or more bonding elements, in particular amino acids, to facilitate the coupling reactions between the fragment of membrane protein and the antigen or hapten.

The covalent coupling of the antigen or hapten according to the invention can be carried out at the Nor C-terminal end of the fragment of the membrane protein according to the invention. The bifunctional reagents which allow this coupling can be determined as a function of the end of the fragment of the membrane protein which is chosen to perform the coupling, and of the nature of the antigen or hapten to be coupled.

WO 99/49892 5 PCT/FR99/00703 These coupling techniques are well known to persons skilled in the art.

The conjugates derived from the coupling of peptides to at least one fragment of an enterobacteria membrane protein OmpA or of a membrane protein of Klebsiella can be prepared by genetic recombination.

The hybrid protein (conjugate) can in fact be produced by recombinant DNA techniques, by insertion or addition of a sequence encoding the antigenic or hapten peptide(s) into or to the DNA sequence encoding the fragment of membrane protein. These techniques for preparing hybrid protein by genetic recombination are well known to persons skilled in the art (cf. for example S.C. MAKRIDES, 1996, Microbiologicals Reviews, 60, 3, 512-538) and will not be developed in the present description.

Thus, the invention also comprises the use, according to the invention, characterized in that the hybrid protein, obtained after coupling between the fragment of a membrane protein and the antigen or hapten, protein in nature, is prepared by genetic recombination.

The Applicant has also shown that, in the absence, of sensitization to Klebsiella pneumoniae, the nasal administration of a hapten coupled to at least one fragment of a membrane protein, such as the protein, in the absence of adjuvant, induced an antihapten antibody response.

The invention relates to the use, according to the invention, characterized in that the pharmaceutical composition contains a fragment of a membrane protein coupled to an antigen or hapten according to the invention, or a transformed host cell which is capable of expressing a hybrid recombinant protein containing a fragment of membrane protein coupled to the antigen or hapten according to the invention, in particular in the absence of adjuvant. Among the transformed host cells which are capable of expressing said hybrid protein, gram-negative bacteria such as Klebsiella pneumoniae,

D

WO 99/49892 6 PCT/FR99/00703 Escherichia coli type K12 currently used in fermentation, or E. coli transformed with an expression vector plasmid containing a strong promoter such as the operon of the tryptophan promoter (trp) are preferred.

Also preferred are gram-positive bacteria such as the nonpathogenic staphylococci, S. carnosus and S. xylosus, since these bacteria do not produce any LPS (lipopolysaccharides) at the membrane surface. These staphylococci can be transfected with expression vectors containing promoters such as trp, or the secretion signal of lipase or even the secretion signal of protein A, or alternatively the signal of the promoter of OmpA of Klebsiella pneumoniae.

Finally, the invention relates to a method for preparing a protein or one of its fragments by the recombinant pathway, characterized in that the protein or its fragment is, after extraction, renatured in the presence of a solution containing a detergent chosen from Zwittergent 3-14, Zwittergent 3-12 and octylglucopyranoside, and in that said recombinant protein is not interferon p.

Preferably, said protein is an enterobacterium membrane protein, in particular of OmpA type. Very preferably, said protein is an OmpA of Klebsiella pneumoniae.

In the method according to the invention, the Zwittergent 3-14 will be preferably at a concentration of between 0.05% and more preferably close to 0.1%.

The following examples are intended to illustrate the invention without in any way limiting the scope thereof.

In these examples, reference will be made to the following figures: Figures 1A and 1B: Analysis by SDS-PAGE electrophoresis of the rP40 protein after purification.

Figure 1A: detection with Coomassie blue lane 1: batch 1, 2 pg lane 2: batch 1, 10 pg lane 3: batch 2, 2 pg WO 99/49892 7 PCT/FR99/00703 lane 4: batch 2, 10 pg lane 5: batch 3, 2 pg lane 6: batch 3, 10 pg Figure 1B: immunoblot and detection with the aid of an anti-P40 rabbit polyclonal serum std: molecular mass standard lane 1: denatured rP40, 100 ng lane 2: native rP40, 100 ng.

Figure 2: Division of the patients according to the O.D. (Optical Density) corresponding to the antibodies, measured by ELISA.

Figure 3: Anti-G1' antibody response.

Figure 4: Anti-rP40 antibody response.

Figure 5: Anti-Gl' IgA-type antibody response.

Figure 6: Isotyping of the anti-Gl' immunoglobulins obtained in secondary response.

Figure 7: Isotyping of the anti-Gl' immunoglobulins obtained in tertiary response.

Figure 8: Anti-G1' total IgG-type serum antibody response.

Figure 9: Isotyping of the serum anti-G1' immunoglobulins after three immunizations.

Figure 10: Isotyping of the anti-G1' immunoglobulins from broncho-alveolar washes after three immunizations.

Example 1: Cloning of Cloning of the rP40 gene: The gene encoding rP40 was obtained by amplification by PCR (Polymerase Chain Reaction) from the chromosomal DNA of the Klebsiella pneumoniae IP 1145 strain (described in patent WO 96/14415). After identification by DNA sequencing, the fragment corresponding to rP40 is cloned into diverse expression vectors, in particular the one under the control of the trp operon promoter, upstream of 9 amino acids of the leader peptide (MKAIFVLNA). The peptide sequence of is represented in the sequence listing by the sequence SEQ ID No 1. In various E.coli K12 strains, the rP40 protein is produced in the form of inclusion WO 99/49892 8 PCT/FR99/00703 bodies with a considerable yield 10%, g proteins/g of biosolids).

Fermentation of rP40 fusion proteins: E. coli K12 transformed with the plasmid pvaLP40 is inoculated in an Erlenmeyer flask containing 250 ml of TSB (Tryptic Soy Broth, Difco) medium containing ampicillin (100 pg/ml, Sigma) and tetracycline (8 pg/ml, Sigma). This is incubated for 16 hours at To 37 0 C with stirring. 200 ml of this culture are inoculated in a fermenter (CHEMAP CF3000, ALFA LAVAL) containing 2 liters of culture medium. The medium contains 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; thiamine, 0.07; magnesium sulfate, 1 and 1 ml/l of trace element solution and 0.65 ml/l of vitamin solution. The parameters which are controlled during the fermentation are: pH, stirring, temperature, degree of oxygenation, supply of combined sources (glycerol or glucose). The pH is regulated at 7.0. The temperature is fixed at 37 0 C. The growth is controlled by supplying with glycerol at a constant flow rate (12 ml/h) so as to maintain the dissolved oxygen tension signal at 30%. When the turbidity of the culture (measured at 580 nm) reaches the value of 80 (after approximately 24 hours of culture), protein production is induced by adding indole acrylic acid (IAA) to a final concentration of 25 mg/l. Approximately 4 hours after induction, the cells are harvested by centrifugation.

The amount of biomass obtained is approximately 200 g, expressed as wet biomass.

Example 2: Extraction and purification of Materials and methods Extraction of After centrifugation of the culture medium (4000 rpm, 10 min, 40C), the cells are resuspended in a mM Tris-HCl buffer, pH 8.5. A treatment with WO 99/49892 9 PCT/FR99/00703 lysozyme (0.5 g/l, 1 hour/room temperature/gentle stirring) allows the release of the inclusion bodies.

The pellet of inclusion bodies obtained by centrifugation (25 min at 10,000 g at 4 0 C) is taken up in a 25 mM Tris-HCl buffer, pH 8.5, containing 5 mM MgCl 2 and then centrifuged (15 min at 10,000 g) The denaturation of the protein is obtained by incubating the inclusion bodies at 37 0 C for 2 hours in a 25 mM Tris-HCl buffer, pH 8.5, containing 7 M urea (denaturing agent) and 10 mM dithiothreitol (reduction of disulfide bridges). A centrifugation (15 min at 10,000 g) makes it possible to remove the insoluble portion of the inclusion bodies.

After dilution with 13 volumes of a 25 mM Tris- HC1 buffer, pH 8.5, containing NaCI (8.76 g/l) and Zwittergent 3-14 the mixture is left to stand overnight at room temperature with stirring, in contact with the air (renaturation of the protein by dilution and reoxidation of the disulfide bridges).

Purification of the rP40 protein Anion exchange chromatography step.

After another centrifugation, the sample is dialyzed against a 25 mM Tris-HCl buffer, pH containing 0.1% Zwittergent 3-14 (100 volumes of buffer) overnight at 4 0

C.

The dialyzate is loaded onto a column containing a support of strong anion exchanger type (Biorad Macro Prep High Q gel), which is equilibrated in the buffer described above at a linear flow rate of 15 cm/h. The proteins are detected at 280 nm. The protein is eluted, with a linear flow rate of 60 cm/h for an NaCl concentration of 0.6 M, in the 25 mM Tris/HCl buffer, pH 8.5; 0.1% Zwittergent 3-14.

Cation exchange chromatography step.

The fractions containing the rP40 protein are pooled and concentrated by ultrafiltration with the aid of an Amicon cell system with stirring used with a Diaflo membrane of type YM10 (cutoff threshold 10 kDa) for volumes of about 100 ml, or with the aid of a WO 99/49892 10 PCT/FR99/00703 Millipore Minitan tangential flow filtration system used with membrane plates having a cutoff threshold of kDa, for larger volumes. The fraction thus concentrated is dialyzed overnight at 4°C against a 20 mM citrate buffer, pH 3.0, containing 0.1% of Zwittergent 3-14.

The dialysate is loaded onto a column containing a support of strong cation exchanger type (Biorad Macro Prep High S gel), which is equilibrated in the 20 mM citrate buffer, pH 3.0, containing 0.1% of Zwittergent 3-14. The rP40 protein is eluted (rate 61 cm/h) for a 0.7 M NaCI concentration. The fractions containing the rP40 are pooled and concentrated as described above.

Results Starting from a 1 liter culture, one denaturation/renaturation cycle makes it possible to obtain 300 mg of protein (estimation by assay according to the Lowry method). 75 mg of rP40 are purified after the two chromatographic steps.

As above, the rP40 protein is concentrated after purification in order to attain a final concentration of between 5 and 10 mg/ml. The electrophoretic profiles show a degree of purity of about 95% (Figure 1A). After immunoblot, the protein is specifically recognized by an anti-natural monoclonal antibody obtained in mice (Figure 1B).

The condition of the protein is monitored by SDS-PAGE. Depending on its form, denatured or native, the P40 protein extracted from the, membrane of Klebsiella pneumoniae has a characteristic electrophoretic behavior (migration). The native form (P-sheet structure) in fact has a lower molecular mass than the denatured form (a-helix structure) under the action of a denaturing agent, such as urea or guanidine hydrochloride, or with heating to 1000C in the presence of SDS (Figure 1B). The rP40 protein is not correctly renatured at the end of renaturation, regardless of whether this is carried out in the presence or absence WO 99/49892 11 PCT/FR99/00703 of 0.1% Zwittergent 3-14. Conversely, total renaturation is obtained after dialysis against a 25 mM Tris/HCl buffer, pH 8.5, containing 0.1% (w/v) Zwittergent 3-14. However, it should be noted that this renaturation is only obtained when the dilution step and the treatment at room temperature are themselves carried out in the presence of Zwittergent 3-14 (negative results in the absence of detergent).

Example 3: Coupling of the Gl' peptide to Materials and methods The Gl' peptide is a synthetic peptide of amino acids, the sequence of which is as follows (SEQ ID No 74): Without the Cys (cysteine) residue added in the C-terminal position, this peptide (portion 1-14) corresponds to portion 174-187 of the protein G of the respiratory syncytial virus, and has, with respect to the native peptide, two major modifications which are: the replacement of the Cys residue at position 13 with a Ser (serine) residue, the replacement at positions 3 and 9 of the Cys residues, which form a disulfide bridge, with, respectively, Asp (aspartic acid) and Orn (ornithine) residues which form a lactam-type bridge.

These modifications are introduced for the purpose of removing the Cys residues of the native peptide in order to be able to carry out a one-to-one coupling of the latter to the protein via the Cys residue introduced in the C-terminal position, while at the same time maintaining the structure of the peptide with the aid of the introduction of a lactam bridge.

The coupling of the peptide to the protein is carried out using the BHA or bromo-N-hydroxysuccinimide acetate reagent (Svenson et al., 1990, Proc. Natl.

Acad. Sci. USA 87, 1347, Bernatowicz and Matsueda, 1986, Anal. Biochem. 155, 95). This heterobifunctional reagent allows activation of the Lys (lysine) residues of the protein by bromoacetylation, and then coupling WO 99/49892 12 PCT/FR99/00703 of the peptide via the free thiol group carried by the Cys residue.

Firstly, the rP40 protein is activated with the BHA. The rP40 is dialyzed against a 0.1 M phosphate buffer, pH 7, containing 0.1% Zwittergent 3-14, for 24 hours at +4 0 C. After dialysis, the concentration is adjusted to 5 mg/ml with the aid of the same buffer, before adding BHA in a proportion of 1.2 mg (50 pl)/mg of The whole is placed in the dark for one hour with stirring and at room temperature.

The activated rP40 is then desalified by gel filtration chromatography (elution with the abovementioned buffer). The fractions containing the bromoacetylated protein are pooled.

For the coupling, the peptide (10 mg/ml in 0.1 M phosphate buffer, pH 7, containing 0.1% Zwittergent 3-14) is added to the activated protein in a proportion of 0.4 mg/mg of protein. After saturation under a nitrogen stream, the tube is again placed in the dark for 2 hours with stirring and at room temperature.

The unbound peptide can be removed with the aid of a dialysis step or of molecular sieve chromatography.

Results The conjugate obtained is characterized by protein assay (BCA or LOWRY method) and by SDS-PAGE electrophoresis. The degree of coupling of the peptide to the protein is estimated by carboxymethylcysteine residue assay: the assaying of the amino acids released by hydrolysis (6N HC1) is performed by HPLC after derivatization with the aid of PITC (Pico-Tag method, Waters).

The degree of coupling determined by this method is approximately 10 Gl' peptides/mole of WO 99/49892 13 PCT/FR99/00703 Example 4: Natural immunity in adults Human sera derived from a clinical study are analyzed by ELISA assay in order to determine the presence of anti-P40 antibodies.

The results are represented in Figure 2.

Among 113 sera tested after 400-fold dilution, 110 sera give a colorimetric signal revealing the anti- IgGs. There are circulating anti-P40 antibodies in all the patients, with levels which are more or less high depending on the patient under consideration.

Example 5: Anti-Gl' antibody response after sensitizations and frequent immunizations BALB/c mice were or were not sensitized twice with a Klebsiella pneumoniae 1145 strain, in order to reproduce the seropositivity found in humans. The mice are subsequently immunized nasally in the absence of adjuvant 7 days after the sensitization. This immunization is carried out with a small amount of antigen, the mice receiving 10 pg of Gl' equivalent coupled to rP40. The mice receive a booster 10 and days after the first immunization. A retro-orbital sinus puncture is performed on the mice 9 days after the first immunization and 10 days after each booster (secondary and tertiary responses). The serum anti-Gl' (Figure 3) and anti-carrier (Figure 4) antibodies are assayed by the ELISA method.

5.1 Assaying of anti-Gl' serum IgGs The results are represented in Figure 3.

In the primary response, the mice presensitized with Klebsiella pneumoniae and immunized with rP40-G1' are the only ones to produce anti-Gl' antibodies.

The level of anti-Gl' antibodies found in the mice presensitized with Klebsiella pneumoniae and immunized with rP40-Gl' is increased after a second immunization. In the absence of presensitization, a second immunization in the presence of the conjugates induces an anti-G1' antibody response.

I

WO 99/49892 14 PCT/FR99/00703 After three immunizations, the anti-Gl' antibody response is increased in the presensitized and non-presensitized mice.

5.2 Assaying of anti-rP40 serum IgGs The results are represented in Figure 4.

The anti-P40 antibody response shows that the mice were sensitized to Klebsiella pneumoniae in identical manner whatever the batch under consideration.

The immunization in the presence of rP40-G1' conjugates slightly increases the anti-rP40 antibody response.

5.3 Assaying of anti-Gl' serum IgAs Secondly, we assayed the serum IgA-type anti- Gl' antibody response: immunoglobulin characteristic of immunizations carried out via the mucous membrane (nasal or oral) pathways.

The results are represented in Figure After a single immunization, IgAs are not detected. After two immunizations, anti-Gl' IgAs are detected essentially in mice presensitized to Klebsiella pneumoniae and immunized with rP40-Gl'. This response is increased by the third immunization. In the absence of sensitization, anti-Gl' IgAs are detected in mice after two immunizations with rP40-Gl' conjugates.

This level of IgA is increased by the third immunization.

5.4 Isotyping of anti-Gl' serum immunoglobulins Two types of response can be observed, Thl and Th2. These responses differ by the profile of cytokines produced and by their functions in the immune response.

IgGls are characteristic of a response of type Th2, and IgG2as are characteristic of a Thl response.

A mixed Thl and Th2 response profile is found only in the mice immunized with the rP40-G1' conjugates, whether or not they are presensitized with Klebsiella pneumoniae (Figure 6).

WO 99/49892 15 PCT/FR99/00703 After three immunizations (Figure the profile remains mixed in the mice immunized with the rP40-G1' conjugates.

Example 6: Anti-G1' antibody response after sensitizations and infrequent immunizations.

With respect to the above protocol, the first immunization is separated from the final sensitization by a period of 3 weeks instead of one week. The anti- Gl' antibodies are assayed in the sera, and, in the tertiary response, in broncho-alveolar washes, by the ELISA method.

6.1 Assaying of anti-G1' serum IgGs As seen in Figure 8, 7 days after the first immunization, anti-Gl' serum antibodies of type total IgG are detected in the mice presensitized to Klebsiella pneumoniae and immunized in the presence of the rP40-G1' conjugates. This antibody response is increased by the two other immunizations.

6.2 Isotyping of serum immunoglobulins The results are represented in Figure 9.

In this case, we also observe a mixed response, we obtain in fact the same titer for IgGl as for IgG2a (Figure In addition, a high level of IgA is found in the mice presensitized to Klebsiella pneumoniae and immunized three weeks later in the presence of the rP40-G1' conjugates.

6.3 Isotyping of immunoglobulins from broncho-alveolar washes In the broncho-alveolar washes, the 4 types of immunoglobulin are found only in the mice sensitized to Klebsiella pneumoniae and immunized 3 times in the presence of the rP40-G1' conjugates (Figure 05-JUN-2003 18:16 I P. 11/14 It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" and grammatical variations thereof, is used in the sense of "including" i.e. the features specified may be associated with further features in various embodiments of the invention.

0 9• COMS ID No: SMBI-00283893 Received by IP Australia: Time 18:15 Date 2003-06-05 EDITORIAL NOTE APPLICATION NUMBER 29391/99 The following Sequence Listing pages 1 to 27 are part of the description. The claims pages follow on pages "16" to "18".

WO 99/49892 PCTIFR99/00703 SEQUENCE LISTING Information for SEQ ID NO: 1 SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 344 amino acids, 1032 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 1 12 N Vnt Lys Ala ie Phe Val Leu Asn Ala Ala Pro Lys GAAA M=rTIC CMAAT G33 GLT an AAh Asp Asn 7 Irp Tyr Ala Gly My as L Gly frrp S e'n 1r His Asp 7hr AT AAC AX 7M T W ar G= r AAh CMG r 7M 7M Ma mC GC ACT 48 Gly Pie TYr Gly Arn Gly Phe Gan Ai Asn Asn Gly Pro Thr Arg Asn Asp Gin G3r TIC 7C CGT AAC 03T TIC CG AAC AAC MAC oWr C0 3 A= =AC Ga DG 66 Leu Gly Ala My Ala Phe Gy Gly '7r Gin Val Asn Pro Tyr Leu Gly Pe Mu crr =r G=r= GM aTIC CGT or 7c C GIT AAC CCG W L L G GA 84 Mat Giy Dyr Asp 'rp Leu Gly Akg tet Ala Tyr LYs CMly Ser Val Asp Mn Gy AM Wr M GC 7MD CIU GC Wr A'G 7AT AAA GX GGar AAC C 102 Ala Pe Lys Ala Gin ly Val Mn Leu Mnr Ala Lys Lau My Tyr Pro Ile Thr CG =Z AAA G= W x WCr G CIG ArM AAA Cdo or 7c a3 An Acr 120 Asp Asp Leu Asp lie Tlyr Tr Ag Leu Gly Gy ttt Val Trp krg Ala Asp Ser GAC GAT CIG GAC MC LTr APC AC dO C ox GGX =0 MG 7W r GAC TC 138 Lys Giy Amn Tyr Ala Ser Thr Gly Val Ser Arg Ser Glu His Asp Thr Gly Val AAA Ox Am:C 7c oar Cr Ac X mC IT =C mT p xA MC MC Acr WC IT 156 Ser Pro Val Phe Ala My Gly Val Glu frp Ala Val 7h r kg Asp Ile Ala Thr CM GM 'rr GT r G3 C G GA GA '7M rr CIT ACr Wr @C AC Gr AM 174 Arg Leu Mu Tyr Gin 7*p Val Agn Asn Ile Gly Asp Ala Gly Thr Val Gly Thr alr C=a W. P. 1rG =3 Cr MC A 'MC MC 0X 3 OX ;Cr GIOU AMf 192 kzg Pro Asp Amn Gy Fat Leu Ser Leu Gly Val Ser Tyr Arg he Mly Gin Glu a~r r Cr- o AAC AM C PX cI x r a= =7 C 7 x TIC a r CYGAA 210 Asp Ala Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala Pro Glu Val Ala Thr GAT an aGk GIT GIr wr an ona aw i an ani am @A mk =nG Aoc 228 Lys His Phe Thr Leu IDys Ser Asp Val Leu Phe sn Phe sn Lys Ala Thr Leu AM MC TiC LC C MC 'aCu A C aIr CIcr TIC AC TIC AAC AAA r AIX CG 246 Lys Pro Glu Gly Gin Gin Ala Leu Asp Gin Leu Tr Thr Gin Leu Ser Asn Met AAA CM nA, n O AGG CIG AT C3; CIG W ACT Cr CIG AC AAC TOG 264 Asp Pro Lbs Asp Cy Ser Ala Val Val Leu GAy 1rT 7nr Asp Akg lie Gly Ser GAT CM AAA @AC Wr G r Garr CIG X 'CC ACC GC =X Am 1C;r =1 WO 99/49892 PCT/FR99/00703 282 Gu Ala Tyr Asn Gin Gin Leu Ser Gu Lys Arg Ala GIn Ser Val val Asp 28 CGA GXr 7C MC G AG CIG C G AAA =r Wzr CP 7M GIC G1T GWC.TWc 300 Leu al Ala Lys Gly Ile Pro Ala Gly Lys nle Ser Ala Arg Gly Mt Gly Glu CIG GT r AA WC A7C CM C r WC AAAlTC 7TE COC Gor AM Wr GAA 318 Ser Asn Pro Val Mw Gly Ams Th Cys Asp Asn Val Lys Ala Arg Ala Ala Leu MOC AAC C GIT ACr G C Wn Gc AAC G M AAA G0 r or c r o C 336 Ile Asp Cys Leu Ala Pro Asp krg krg Val Giu Ile Glu Val lys Gly Tyr Lys ATC GT 7M CM Mr GA3 WrT a r G CAG AC QA IT MA OG Mc AM 344 Glu Val Val Thr Gin Pro Gin Ala AA IT GM Ar M Cr CG G= Information for SEQ ID NO: 2 G2A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N Thr Val Lys thr Lys Am Thr 1-r Thr Thr Gin Thr Gin Pro Ser L5 Pro Thr hy 4s AM GIG AA AMA M A= AM A= A= CAS P=A AA= AAP 150 GIn Arg GIn Asn Lys Pro Pro Asn Lys Pro Asn Asn Asp Phe- His ERe G.u Val Phe Asn Phe 01G Mr APC AAA CM CcG APC AAA OX AP AAC G A G7G TM APC 77C 17 173 176 182 186 Val Pro Cys Ser Lie Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn GIG CW AX ATC PAM C APC CM A=3 P7M C 3C A A a(r MC 033 MC 192 Lys Lys Pro Gly Lys Lys Thr Tr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr Thr Lys AAh A ax; G= AAA AAA A= AM A=Z AAA C2 PO MA AAA cPm AM APA P= Pm AAA 213 230 Lys Asp His Lys Pro Gin Thr Mtr ys Pro Lys Glu Val Pro Mr hr Lys Pro C AAA GIC GO AP M; OG PAM PX AAA 02G AAA CPA GIG rr- AMC= A A a- 3' Information for SEQ ID NO: 3 G2B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 3 PCT/FR99/00703 130 N 7TV Ala Gln Thr Lys Gly Arg Ile Thr Thr Ser 7VW Gin Thr Asn Lys Pro Ser Thr Lys AXCPO G0 VG AMA al: Cr AIC AX A AMC X= UPLOG AX APC A M AM AX AAA 150 Ser Arg Ser Lys Mn Pro Pro Lys Lys Pzo Lys Asp Asp Tyr His Re Glu Val Phe Asn We Am aIr AX AAA APC OX; AM AAAM APA UX ZV WC OC TIC QA GIG TiC PlC TIC 171 173 176 182 186 Val Pro Cys Ser Ile Cys Gly Asn Asn Gin Leu Cys Lys Ser Ile Cys Lys Thr Ile pR Sar GIM PM AM G= APC AlC CIG CIG 7a: AAA AGC AC 7 M AM 192 Mn Lys Pro Lys Lys Lys Pro 71r Ile Lys Pro Thr Asn Lys Pro Thr Thr Lys Th Thr Asn A AM AS AA C AM C C 7 MC AM CC G ArC AAA 0M 0 TM IM 1 M IA J 213 230 Lys Arg Ap Pro Lys 1hr Pro Ala Lys Wt Pro Lys Lys Glu Ile Ile Thr Mn C AAA C3T CPT COS AT AM aX a; APA A17 C AFG NIG GM AC AI PX APC 3' Information for SEQ ID NO: 4 G2A8Cys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys Pro Thr Thr Lys A= GIG AAA AM AAA AAC AX AM Xr- A= CG P= APA C AX P= AMA 150 Gin Arg Gin Am Lys Pro Pro An Lys Pro Mn Asn Asp Pe His he Glu Val Re n R-e CPG =r DG AAC MA AIc C= APC AA CM APC PC GT TIC CI CC @A GMG TIC PC TIC 171 173 176 182 186 Val Pro Ser Ser Ile Cys Ser Amn Amn Pro hr Cys Trp Ala Ile Ser Ls Arg Ile Pro As GIG CM A= PAXA= AX AC APC A= 7= 7W GOS AIC X0 AAA 03r A= CM APC 192 Lys Lys Pro Giy Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Rhe Lys Thr Thr Lys AAA A CCG G AG A AAA A= PM AX AAA C=3 AAA AAA CM TIC AAA AM PX APA 213 230 Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val Pro Tnr Thr Ls Pro C MAA GW CT AAA MG C A= AM APA AA A GM a=C PC A 3' Information for SEQ ID NO: 5 G2B8Cys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 PCT/FR99/00703 130 N Thr Ala Gin Thr Lys Gly Arg ne Thr Thr Se t Gin thr Asn Lys Pro Ser 7T Lys AX an UG MAA AA GM 0r A7 ACE PX AM OQ G. AM AC AAA CM AX AAA 150 Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys Ap Asp Tyr His RE Glu Val Pe Asn Re AM 3r A APA APC C5 Ca; AAAMAPA ax AAA GT GM TPC TC @A G1r TIC APC TIC 171 173 176 182 186 Val Pro Ser Ser le Cys Gly Asn Asn Gin Leu Cys Lys Ser le Ser Lys Thr le Pro Ser GD 3 P0 C AMAMGM A APC OGC M 7AA AM a AM AA AC M CJ5 AM 192 Asn Lys Pmo Ls Lys Ls Pro Thr Ile Ls Pro Thr Asn Lys Pro hr Thr Lys Thr Thr Asn APC AAA ACOGAA APG A G AMC AA C 0 ArC AAA am Pi =cRC AM X APC 213 230 Lys Arg As Pro Lys Thr Pro Ala Lys Mat Pro Ls Lys Glu Ile Ile Thr Asn c AAA O1 G;C O M AA PC a; MPA MG Oa NG AC G*M MC A7C ;M AMC 3' Information for SEQ ID NO: 6 GlACys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ile Cys Ser As n sn Pxo Thr Cys Trp Ala Ile Cys Lys C AX A2C IC X AC AAC CM 'AIC GM AM AAA 3' Information for SEQ ID NO: 7 G1BCys SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE, peptide 174 176 182 186 187 N Ser lle Cys Gy Asn Asn Can Leu Cs Lys Ser Ie Cys Lys C AC AC =I AAC AAC a:e CIU 'IC AAA A AAA 3 Information for SEQ ID NO: 8 GlA SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide WO 99/49892 5 PCT/FR99/00703 174 176 182 186 187 N Ser Ile cys Ser Asn Asn Pro Ihr Cys Tzp Ala Ile Ser lys C ACATC TGC P AC AAC C- A;C 7IC 7WX GM AIC AGC AAA 3' Information for SEQ ID NO: 9 GIB SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Ser Lys C AC ATC C GC AAC AAC oCa CIG DC AAA C AC AGC AAA 3' Information for SEQ ID NO: 10 G1'A SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Cys Lys C Information for SEQ ID NO: 11 Gl'B SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ile Asp Gly Asn Asn Gn Leu Orn Lys Ser Ile Cys Lys C Information for SEQ ID NO: 12 G1'A6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide Wo 99/49892 PCT/FR99/00703 174 176 182 186 187 N Ser e Asp S Asn Asn Pro M= On 1)p Ala ie Serts C Information for SEQ ID NO: 13 G1'B8C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ile Asp G As An Gn Lu Orn lys Ser le Ser ys- C Information for SEQ ID NO: 14 G2A6CF SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N Thr Val Lys Thr Ls Am t Thr Thr Thr GIn Thr GIn Pro Ser Ls Pro Thr Thr Lys A= GIG AA A= AAA ArC Am AM E AM NE U s ZA AM =E A=X= AAA 150 163 165 168 170 Gin ArL Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn AsS5e His Ser Glu Val ser Asn Ser AG Cr (UG APC APA CM; AC MA al APC APC GAT CAT =I Gp GIG =s N r 171 173 176 182 186 Val Pro Ser Ser Ile Cys Sr Asn Asn Pro Thr Cys vp Ala Ile Scr Lys Axrj Ile pro Asn GIG OX PM P AC I A ATC AFC Ca Xn MG am P= N im ar ic ax; Nc 192 Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro R Lys Lys Pro Thr RPh Lys 'h11 Thr LS AA MA COS G= AAA AP ANE= N AxA ax A AA AA c33 NE 17C Am PE AAA 213 230 Lys Asp His Ls Pro Gin Th Thr Lys Pro Lys Glu Val Pro Thr thr Lys Pro C AA GV CAT AAA CCG NE A= AM AAA CCG AP Gm M AGXA G N A 3' Information for SEQ ID NO: 15 G4A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 .176 182 186 187 N Val Pro Cys Ser Ile Cys Ser Asn Asn Pro 7hr cys Trp Ala le Cys Lys C GIG CaI 7C Xr AC =IAX AAC MC an AC 7ID GM 1= AAA 3' WO 99/49892 7 PCT/FR99/00703 Information for SEQ ID NO: 16 G4A8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys C GIG C AGC ~oC ATC TGC AGC AAC AAC 033 PCC TOC T7 G C AC AAA 3' Information for SEQ ID NO: 17 G4B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Cys Ser lie Cys Gly Asn As n Gn eu Cys Lys Ser Ile Cys Lys C -GIG CC TC AC ATC xGC GC AAC AAC OG CIG ?XC AAA AC ATC T'C AAA 3' Information for SEQ ID NO: 18 G4B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Ile Cys Gly As Asn Gin Leu CLys Sr e Lys e Lys C -GIG 00 AGC PC ATC TGC GC AAC AAC CA CIG TGC AAA AGC ATC AGC AAA 3' Information for SEQ ID NO: 19 G4'A SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Asp Ser Ile Asp Ser Asn Asn Pro Thr Cm Trp Ala Ile Orn Lys C WO 99/49892 8 PCT/FR99/00703 Information for SEQ ID NO: 20 G4'A6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Ser Lys C Information for SEQ ID NO: 21 G4'B SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Asp Ser lie Asp GLy Asn Asn Gn Leu Orn Lys Ser Ile Orn Lys C Information for SEQ ID NO: 22 G4'B6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Ile Asp Gly Asn Asn Gin Leu On Lys Ser Ile Ser Lys C Information for SEQ ID NO: 23 G200A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein Wo 99/49892 PCT/FR99/00703 140 N Gln Thr Gln P=o Ser Lys Pro 2br Thr Lys Gn Arg Gn-Asn Lys Pro Pro Asn MC Ar CG 03AX AAA CM AM ACE AAA c rG ar00G AAC AAA CMl C= AAC 158 173 176 Lys Pro Asn Asn Asp Pe His PE e Giu Val Phe Asn Rle Val Pro Ser le Cys AAA AAC AAC GT TIC C TIC GAA GIG =C AAC =IC GIG CM1W AX AIC 7M 177 152 186 Ser Asr Asn Pro hr Cys Trp Ala Ile Cys ys Arg Ile Pro As ns 1's Pro Gly AW AAC AAC C33 AX 7W 033MC 7W AAA 03~ A7C CM AAC AAA AAA CM a= 196 200 Lys Lys Mhr flr -nr C AAA AAA PAC AM AX 3' Information for SEQ ID NO: 24 G198A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin Mr Gin Pro Ser Lys Pro Thr Thr Lys Gln Arg Gin Asn Lys Pro Pro An 51- CAG AC CAG AX 0 AX AA AM AX MA G 0 CAAC AAA C mAAC 158 173 176 Lys Pro Asn An Asp Pe His Re Glu Val Phe Awi P- Val Pro Cys Ser le Cys AAA COOAAC AC GA TIC T TIC AA GIG TIG C TI GIG AX ATC 7 177 182 186 Ser Asn An Pro Thr Cs Trp Ala le Cs Lys Arg Ile Pro As 14Ls Lys Pro Gly AC AAC AAC 3 ACC 7a; MC 7 MA wr AC C A AAA cm w 196 198 Lys Lys Thr C AAA AAA AX 3' Information for SEQ ID NO: 25 G196A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gln Thr GIn pro Ser Lys Pro Thr Thr Lys Gln Arg Gin Asn Lys Pro Pro Asn C0 AX CG 0 AOC AAA AM Aoc AA c00 =0G AAC AAA C03 C AAC 158 173 176 Lys Pro Awn Amn Asp Phe His FRe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys MAA OM AAC AAC GA TIC TIC A GG TIC AC TIC GIMG 7AIGC AC 7W3Z 177 182 186 Ser Asn Asn Pro Mir Cys Trp Ala Ile Cs Lys kg lie Pro Asn s Lys Pro Gly AW AAC AAC C3 ACX W 7M GM=7 AAA 3 MC AAC AAA MA CI3 G3C 196 Lys C AAA 3' WO 99/49892 10 PCT/FR99/00703 Information for SEQ ID NO: 26 G194A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin Thr Gin Pro Ser Lys Pro 'Dir Mr Jys Gn Arg Gin An Is Pro Pro Asn -PD AM MG AX Mh OM ACC AM AAA CFG ar CW AC AAA C03 003AAC' 158 173 176 Lys ProAm Am Asp P His Me Glu Val PIhe Asn Phe Val Pro Cys Ser Ile Cys AAA a. AAC AAC GAT TC CT @A GIG T C TIC GIG 7W AGC A7C 7W 177 182 186 194 Ser A As Pro 7hr Cys Trp Ala Ile Cys Lys Arg Ile Pro An Lys Lys Pro C AGC AAC AAC C M IA =D 7M aC A= 7WA =r A= AAC AAA AAA 3' Information for SEQ ID NO: 27 G192A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 52 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin Thr Gin Pro Ser Lys Pro hr Thr Lys Gin Arg Gin Arn Lys Pro Pro Asn CG A= Ca3 X;C APA AC A= AAA CG C MC AAA CM MC 158 173 176 Lys Pro Asn Asn Asp Phe His Pie Gu Val Phe An Phe Val Pro Cys Ser Ile Cys MA =G AC MC GAT Tic cAT mA GIG TIc AAC Tc GIG o 37 Am=C' 7w 177 182 186 192 Ser Asn Asn Pro Thr Cys frp Ala Ile Cys Lys krg Ile Pro Asn Lys C AXZ AAC MC 'M 1 GM A'C MA CIG A= AAC AAA 3' Information for SEQ ID NO: 28 G6A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin h Gin Pro Ser Lys Pro Thr mhr Lys Gin Arg Gin Asn Ls Pro Pro Am O ACC CPacZ P0r AA CZ; Acr Am AAA cAG cur G mC MA cm =AC 158 173 176 Lys Pro Am Asn Asp Phe His Phe Glu Val Phe Asn Re Val Pro Cys Ser Ile Cys AAA C3 MAC AAC TIC =T G A GIG TIC MC ATC GIG an j A: Ar 7W WO 99/49892 11 PCT/FR99/00703 177 182 186 190 Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro C AM AAC AAC ACPC TC =3 GO AIC 1TW AAA CT AaC M 3' Information for SEQ ID NO: 29 G7A SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 N Lys Pro Asn Asn Asp. he His Phe Glu Val Phe Asn Phe Val Pro Cys Ser le AAA 03 AA C AAC TIC Or TIC GA GIG TIC AAC TIC GG TC C AC 176 182 186 190 Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro C TG C AAC AAC G=3 AC 1'W AA =T ATC COO 3' Information for SEQ ID NO: 30 G200A8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gln Thr Gin Pro Ser Lys Pro Thr hr iLys Gn Arg Gin Asn Lys Pro Pro Asn (CA Ap= CG C AC AAA C AM AM AAA M CaT aG AAC AAA cm AAC 158 173 176 Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys AAA AAC AAC GAT TIC T TIC A GIG AAC TC GG C AC ATC O 177 182 186 ser Asn Asn Pro Thr Cys TLp Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys Pro Gly AXc AAC AOC ac7 L A= X a e AAA Cr ArCe Oa AAC AAA AAA C 3co 196 200 Lys Lys Thr Ihr Thr C AAA AAA AX AD AX 31 Information for SEQ ID NO: 31 G198A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 -12 PCT/FR99/00703 140 N Gin Thr in Pro Ser lys Pro Mr hr IVs Gin Arg Gin Am Lys Pro Pro Am QPG AC CG ADC AAA CM AM AM AAA CG Cr aG AC AA CM CM AAC 158 173 176 L's Pro An Asn Asp Pb His Pe Mu Val Phe sn Pe Val Pro Ser Ser fle Cys AAA OM XC C GAT TIC CAT TIC CA GG TIC AC TC GMTC AGC AC A= 177 182 186 Ser mn mn Pro thr Cys 7rp Ala Ile Ser Lys Arg lIe Pro Asn Lys Lys Pro Gly AOC AAC AAC AC A u 7X i Cl ATC Mr AAA CGT ATC CCC AAC AAA AAA C= W 196 198 Lys Lys hr C AAA AAA ACC 3' Information for SEQ ID NO: 32 G196A8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin Thr Gin Pro Ser Lys Pro 7hr Thr Lys Gin Akg Gin Am Lys Pro Pro Asn MG A= OG AX AAA Ct X= MAA CAG OG AAC AAA AAC 158 173 176 Lys Pro Asn Asn Asp Phe His FRe Giu Val Pbe Asn Phe Val Pro Ser Ser Ile Cys AAA AAC MAC GAT I CA TIC A GI TIC AAC'TI GIMt; AM AG= AC 'G 177 182 186 Ser Asn Asn Pro 7fr Cys Trp Ala Ile Ser Lys Axg Ile Pro Asn Ls 14s Pro Gly AM MC MC ACC ATC AX MA AC AAC AAA MAA MC 196 Lys -C AAA -3' Information for SEQ ID NO: 33 G194A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N GIn Mr Gin Pro Ser Lys Pro 7r Thr Lys Gin kzg Gin Asn Lys Pro Pro Asn CAG ACC CAG AM AAA CC ACt AMT AAA CAG =G AAC MA a A C 158 173 176 Lys Pro Asn Asn Asp Phe His Phe Giu Val Phe Mn Phe Val Pro Ser Ser Ile Cys AAA WC AC MAC C TC CAT TIC GhA GIG TI AAC TIC GIG =PX AMZ =M7 177 182 186 194 Ser Asn Asn Pro 'fr Cys 7ip Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys Pro C ASC AAC AAC CZ; AC'IZ Wt Ga AC AXZ MA r AC =MC AA MA Ca 3' WO 99/49892 13 PCT/FR99/00703 Information for SEQ ID NO: 34 G192A8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin hr Gin Pro Ser Lys Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn 51- CAG AX GG PW AM cm AM A A AA CA G =rCG AAC AAA cM AC 158 173 176 LYs Pro Asn Amn Asp Rie His PR Glu Val Fe Asn Phe Val-R P Ser Ser Ile Cys AAA CM AAC A C A TIC C Ti GA GIG TIC AAC TIC GIG C);o Pa AMZ ATC 177 182 186 192 Ser Asn Asn Pro Thr Cys Mfp Ala Ile Ser Lys Arg le Pro m Lys C AM AAC AAC Ca X= 7 AM A A CI C W AAA 3.

Information for SEQ ID NO: 35 G6A8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Gin hi Gin Pro Ser Ls Pro 71r 7tr Lys Gin Arg Gin m Ls Pro Pro Asn CAG ACC CAS033 PX AA P= ACC AA CAG CGT M MC MAA CM AAC 158 173 176 Lys Pro Asn Asn Asp Pe His Phe Glu Val Pe An Phi Val Pro Ser Sex Ile Cys AAA CM AAC AAC G TIC T TIC A GIG TIC AAC GIG MAM AM A 177 182 186 190 Sex As- Asg Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile Pro C AM MC AC iPa 7M AC AA A= CM 3' Information for SEQ ID NO: 36 G7A6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 N L's Pro Asa As- Asp Phe His Phe Giu Val Phe Asn Phe Val Pro Ser Ser Ile 51- AAA Ca AAC AAC GAT TI C TI= MA GIG TIC AAC TC GIG=; pA )X A7 176 182 186 190 CYs Ser Asn Asn Pro Mr Cs Ip Ala le Ser Lys Ax g Ile Pro C 1C AGC AA C C0 AM MC 7U3 GM A I AM Ir MC 3' WO 99/49892 14 PCT/FR99/00703 Information for SEQ ID NO: 37 G200B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N ser Thr GIn Thr Asn Lys Pro Ser Thr Lys Ser Arg ser Lys Asn Pro Pro Lys Lys Pro A PAX OG AIC NcAAA=; AM A AM AM P AAc ax; CM AA PA CO 160 173 176 Lys Asp Asp Tyr His Phe Glu Val P-e Asn Phe Val Pro Cys Ser lie Cys Gly Asn Asn Gin AAA GAT GAT TPC CPC TIC A GI" TIC AFC T GIM C P MlC 7W G= NC AxC rGG 182 186 200 Lei Cys Lys Ser le Cys Lys Thr Ile Pro Scr Asn Lys Pro Lys Lys Lys Pro Thr le- C CIG 7W AAA Xr- A7C T A PM3 A3C Ca P= ArC AM aX AM NrG AM A7C ?I A- 3 Information for SEQ ID NO: 38 G198B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser Thr Gin Thr Asn Lys Pro Ser Thr Ls Ser Arg Ser Lys Asn Pro Pro Lys Ls Pro PG-- A= CG AM APC PA AA= AAA PX 03r AM AM AP4= =PAP AP AAA COG 160 173 176 Lys Asp Asp Tyr His fPe Glu Val Phe Asn Phe Val Pro CYS Ser Ile Cys Gly Asn Asn Gin AAA GC GT 7C C7 TIc GPA G1 TIC ANC TIC GI 7 AG P C 7W 3- APC AC C.G 182 186 198 Lau Cys Lys Ser Lie Cys Lys Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro C cIG AAAm AC 7w= AnA Pa: AC COG A C M AAA APAA AAA CG 3' Information for SEQ ID NO: 39 G196B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 15 PCT/FR99/00703 140 Set w Gn r Amn Lps Pro Ser Thr Lys Ser frt Ser Lys An Pro Pro Lys Lys Pro AX AX= CC: I=AC AAR CCG P M A AFA AX AM APC= A AA CA 160 173 176 Iys Asp Asp Tyr His Pre Gin Val he Asn he Val Pro Cys Ser Ile Cys Gly Asn Asn Gin APA Gt G;C 7X CIC TIC GPA GIG IC AFC GC= T I )X P: mc A Q APC APC C7G 182 186 196 Lau Cys Lys Ser Ile Cys Lys Thr lie Pro Ser Asn Lys PRo Lys Lys C CIGW AAA AM M U WAAA AMCM AMC AC AAA CM ANA AP 3' Information for SEQ ID NO: 40 G194B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N ser Thx Gin Thr Msn Lys Pro Ser Thr Ly Ser Arg Ser Lys Msn Pro Pro Ls Lys Pro AC PCC Uri ACC AC AA 03 ACE AZ AA AM: 03' PMr: A A APC a XL CAA AAA OOG 160 173 176 Lys AFp Asp Tyr His Rhe Giu Val Phe Asn Phe Val Pro Cy5 ser Ile Cys Gly Asn Asn Gln AAA GC GT TPC CC Tic C Gi Tic AFC =ic GTG C PC 70 A M o GMVC AC crM 182 186 194 LeU Cys Lys Sec Ile C~s Lys Thr Ile Pro 5cr Asn Ly Pro C CIG 7 XAA A= A=C =AAA A= AM A CMC 31 Information for SEQ ID NO: 41 G192B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Secr Thr Gin Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro I P= CG AC APC AA a3 AM PC: AA Xr- AM AAA MPC =CM AAA FAA ar, 160 173 176 Lys Asp Asp Tyr His Ehe Glu Val Ee Asn Phe Val Pro Cys Ser Ile Cys Giy Asn Amn GIn AAA GU GhT TAC CAC TIC GPA GIG TIC AC i GIG 0= A PXC 3: AAC APrC ONG 182 186 192 L- Cys Lys Ser Lie Cys Lys Thr Ile Po Se An C CIM APA AX A=C 7 3 FAA PAC COG PM: AC 31 Wo 99/49892 -16 PCT/FR99/00703 Information for SEQ ID NO: 42 G6B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser Thr Gin 7br Asn Lys pR Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro AM APr- CRC; x= AAC ApA CCG Aa: AMC AMA A= C6 A=X AAA A Y OMm aX A AMAQj 160 173 176 Lys Asp Asp Tyr His re Glu Val Re Asn Re Val Pro Cys Ser lne Cys Gly Asn Asn Gin AAA CAT GC TAC C7C TIC @A G7G T=C APC I GG AX AM 7 G= AC APC CG 182 186 190 Ilm Cys Lys Ser nle Cys Lys Thr lie Pro C CIG TXIAAA AMP X TM APA ACaC ATC C 3' Information for SEQ ID NO: 43 G7B SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Lys Pro Lys Asp Asp Tyr His Re Glu Val phe Asn Re Val Pro Cys Ser Ile Cys Gly AAA AAA GAT GAT vc C c TIC GA GIG TIC C TIC GI C= AM= AC= G= 182 186 190 Asn Asn Gin Leu Cys Lys Ser le Cys Lys thr Ile Pro C APC APC Cr; CIG =AA AM I 1 AAA A= 7C X 3' Information for SEQ ID NO: 44 G200BdC SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser Thr GIn Thr Asn Lys Pro Ser Thr Lys 5c Arg ser Lys Asn Pro Pro Lys Ls Pr AM AXP= C AM A7; PACC PX A AAA AM Mr AAA =O AM 160 173 176 Lys Asp Asp Tyr His Re Giu Val Re Asn Re Val Pro Ser Ser Ile Cys Gly Asn Asn Gin AA GT GAT T C TC A GG TIC= AAC TIC GIG M AM C GM AAC NC CW, 182 186 200 Le Cys Lys ser le Ser Lys Thr Ile Pro Ser Asn Lys Pro Lys Lys Lys Pro Thr Lie- c CIG 7W AAA AM AM P AA P A= COG PM AC AA C AM AP AA X; A= AI- 3' Wo 99/49892 -17 PCT/FR99/00703 Information for SEQ ID NO: 45 G198B5C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser Thr Gin Thr Asn Lys Pro Ser Thr Ls Ser Arg Ser Lys Asn Pro Pro 14s Lys Pro Am Am Ar- PCA ca; ;x Am A m r Pwr Am A Apc an Am AAm 160 173 176 Lys Asp Asp Tyr His Ehe Glu Val he Asn Re Val Pro Ser Ser le Cys Gly Mn Asn Gin AMh G GW Mr XTIC =FA GG TI A TIC GIG AMAMM AI C C;G 182 186 198 Lau Cys Lys Ser Ine Ser Lys Thr Ile Pro Ser Asn 14s Pro .Lys Lys Pro C CIG AAPA PZ AC AS AAA AM AIC CMP A APA C; APA A.G AA Ca; Information for SEQ ID NO: 46 G196B5C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Ser Th Gln Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys pro AYc Pcc cx. X= Ar- AAA Am Ar AAFL A r AMP ApC am Am Am CM 160 173 176 14W Asp Asp Tyr His Re Glu Val Re Asn Re Val Pro ser ser Lie Cys Gly Msn Asn Gin AAA Gv QU 77C CP C TIC GnA GrG TIC MG TIC GM Am Am AM ar_ AAC APC MC G 182 186 196 Le.I Cys Ls Ser Ile Ser Lys Thr Ile Pro Ser Asn Lys Pro Lys Lys C CIG APA POC A= PCC AMA AX PC COG POC ATC AA AA ANG 3' Information for SEQ ID NO: 47 G194B8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 18 PCT/FR99/00703 140 N Ser Thr Gin 7r AM IS- Po Se MV 10 Ser Ag Ser LW Ami Pro PM Ls Ip Pro pACE 2 PG AX AAAA CM AM AMG A AM CA AAA AM CMOX AM AAAn 160 173 176 Lys Asp Asp Tyr His Ehe Glu Val .P Asn FPe Val Pro Sex Ser Ile Cqs Gly Asn Asn Gin AAA G T G C AT C TIC M GAAIG NC TM GIG AX AM C A C APC G 182 186 194 Leu Cys Lys Ser Lie Sex Lys Thr Ile Pro Sex Asn I4ys Pro C CTC AAA AMIC A AAA PAr PAC C0 3 M AFC PM aM 3' Information for SEQ ID NO: 48 G192B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Sex Thr Gin Thr Asn Lys Pro Sex Thr Lys Ser Ag Ser Lys Asn Pro Pro Lys Lys Pro PGX AOU3Gr AA AAA C Mr PC CA A PC 7A AA NC O O AA AA a 160 173 176 Lys Asp Asp Tyr His Phe Glu Val Phe Asn hee Val Po Sex Ser Ile Cys Gly Asn Asn Gin AAA Or G;C 77C CTC TIC QA GIG T= AC TIC GIM =A P; PC 7W AF AF C U 182 186 192 ILeu Cys Lys Ser Ile Ser Lys Thr Ile Pro Sex Asn C CIG AAA i Am Am AAA AX AM 003 PXAA1C 31 Information for SEQ ID NO: 49 G6B6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Sex'Thr Gin Thr Asn Lys Pro Sex Thr Lys Ser Arg Sex Lys Asn Pro Pro Lys Lys Pro PA3 A= cAG Px A c AA oc Pa p AA P =A AP A am X AAA AAA anG 160 173 176 Lys Asp Asp Tyr His hei Glu Val Phe Asn Phe Val Pro Sex Ser Ile Cys Gly Asn Asn Gin AMA GAT G 77C C7C TIC M GIG TIC AFC TI GIG a= Am P05 AC Tw AC NC APCP 182 186 190 Leu CyS Lys Ser Ile Sex Lys Thr Ile Pro C CIG T AAA PC- AIC PX MA AM A7C 02 3' WO 99/49892 19 PCT/FR99/00703 Information for SEQ ID NO: 50 G7B8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Lys P=o Lys Asp Asp Tyr HiS Fhe Glu Val R'e'Asn Phe Val Pto Ser Ser Ile Cya Gly AA A AMA GO GC VC 0C T=C G'A GI TIC APC TIC GIG 03 PAM AM AC= GX 182 186 190 Asn Asn Gin L Cys Lys Ser Ile Ser Ls Thr Ile Pro C APC APC UG CIG W A A AC A2C AX AA AX= AIC- CO 3' Information for SEQ ID NO: 51 G2V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N GIn Asn Arg Lys Ile Lys Gly Gin Ser Thr La Pro Ala Thr Ag Lys Pro Pro Ile Asn CPA AzC APk AAA At IiAA, cr MA 7 CM C AMCM O AT An 150 Pro Ser Gly Ser Ile Pro Pro Giu Asn His Gin Asp Fs Asn Asn Phe Gin Thr Lea Pro Tyr CCR 7. MkAC A7C Ca @a GA APC CAT CAA GC CC A;C APC TIC A AM C e 171 173 176 182 186 Val Pro Cys Ser 7rx Cys Glu Gly Asn Leu Ala Cqs Ser Lau Cys His Ile Glu Thr Glu GT CC= W PG r A cr AA crr GM 1nr 7M 7M CMC W ATr G P0 MGR 192 Arg Ala Pro Se Arg Ala Pro Thr Ile Thr Leu Lys Lys Thr Pro Lys Pro Lys Thr 7br Lys AGA CA CA A r AGA G2k CCA ACA AIC A= CIC A M CA AAA C 02AAA AMCJ AM AM 213 230 Lys Pro Thr Ls Tnr Thr Ile His His Arg Thr Sex Pro GIu Thr Lys Leu Gln c AAGCCA A AP AM ANC p= t GC ACA ACC PAr CCA @A AM AAA CIG CPA 3' Information for SEQ ID NO: 52 G2V8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 101 amino acids, 303 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 130 N GIn Am Arg Lys Ile Lys Gly Gli Ser 1l-r La, Pro Thr Arg Lys Pro Pro Lie Asn CA Ac NA APA iac Am cPA ih z cma Ge m Am wk ccA T Aic WO 99/49892 20 PCT/FR99/00703 150 Pro Ser Gay Ser Ile Pro Pro Glu Asn His Gin Asp His Asn Asn Ph Gin Thr Leu Pro Tyr CCA IC AA PGZ A 0A GA ATC CAT CA GAC OC AM APC CM AM COC TAT 171 173 176 182 186 Val Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Lw Ser 1A. Ser His Lie Giu Thr Glu GIT 0= AM AGzr PEA 7W GA Gr AFW Crr Wk =u TI7M 7M CIC AX CAT AT GrG a G 192 Arg Ala Pro Ser krg Ala Pr= nx lie TRn L1 LYs Lys Thr Pro Lys Pro Lys 7hr Thr Lys AGN cA 00 PM Ak GM POC P M= YcM C1C APA APG 02aaA AAA 02 AM ACA AAA 213 230 Lys Pro hr Lys whr hr Ile His His Arg Thr ser Pro Glu Thr Lys Leu Gin C APG 0)2 PaZ ?pG P AMC O C AM3 A X AX GA AM AAA cIm CAA 3' Information for SEQ ID NO: 53 G200V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N LeA Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser lie Pro Pro Glu Asn His CM 02A GM ACA X AA a7 CC PAATr PAT CCA 7 GM AaC;A C 0 CO A APC Cre 160 173 176 Gin Asp His Asn Asn Phe Gln 7hr Leu Pro Tyr Val Pro Cys Ser lbr Cys Giu Giy Asn Lau CAA GPC C7C APC AC TC PA A CIC CM TWT GIT CM AM' X 7W GP Gr APT crr 182 186 200 Ala Cys Le Ser Leu Cys His Ile Glu Thr Giu Arg Ala Pro 5cr Arg Ala Pro Thr Ile C GM= T TA crm CP; AT G G AMGA A M2 GM PC AM Mk Mk ACA A=C 3' Information for SEQ ID NO: 54 G198V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lei Pro Ala Thr kxg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His CA 02GM AC AG AAA 0a 0A AT AT 02 A 0TM PG AIC 020 C A APC OT 160 173 176 Gin Asp His Asn Asn Phe Gin Tr Leu Pro 'yr Val Pro Cys Ser Thr Cys Giu Gly Asn Leu CAA ICC CTC AFC APC TIC CPA PC CIC QCO TAT CIT or Pk TGI' CPA or AAT CT 182 186 198 Ala Cys Leu Ser Leu Cys His lie Glu Thr Glu Arg Ala Pro 5c Arg Ala Pro C 02 A TG TIA 'CA CI l'a CAT AaT CGR AO3 GAA PLA GM CM P M A GM 2 M 3' WO6 99/49892 -21 PCT/FR99/00703 Information for SEQ ID NO: 55 G196V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leu Pro Ala Thr Arg Lys Pro Pro le Asn Pro r Gly Scr lie Pro Pro Glu Asn His C]M OM GX PcA pG AA oo a TI' A7U T 13 cA 7a mm Am MC am ACM C aoM 160 173 176 Gin Asp His Asn Asn Pe Gin Thr LeA Pro Tyr Val Pro Cys Sex Thr Cys Giu Gly Asn Leu CPA GAC CPC APc APC Trc Cm Pm cic c 7 T AGG ACA r GAA Pw r AIC crr 182 186 196 Ala Cys Lea Ser leu Cys His Ile Glu Thr Glu ?rg Ala Pro Ser Ag C aA T1k 7M C C TGa TIU =G GCA GPA PA GM__MPM Ppk 31 Information for SEQ ID NO: 56 G194V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lea Pro Ala Thr Arg Lys Pro Pro le Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His C7 OM GM AAA ax Ok ATT APX OCA Th W AM PaIC Mk CM MA AP CT 160 173 176 Gin Asp is Asn Asn he Gin Dhr Leu Pro Tyr Val Pro Cys Ser Thr Cys Glu Gly Asn Lea CA CC C APC APC TI CAA P CA C Ty GIT AG_ 1P7 GA G=3r MT crr 182 186 194 Ala Cys Lea Ser Lea Cys His lie Glu thr Glu Arg Ala Po C GM TX TA 1r Tc r AT GG P3 GPA P~k GM C 3' Information for SEQ ID NO: 57 G192V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 22 PCT/FR99/00703 140 N Le Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser aly Ser Lie Pro Pro Giu Asn His CIA CA ar- ACm PGA AAA m kA A~r CA TM GA AM MC 3k k G APC Cie 160 173 176 Gin As His Asn Asn FPe GIn Thr Leu Pro Tyr Val Pro Cys Ser Thr Cys Glu Gly Asn Lai CPA (PCC ACC APC TIC CAh PA C =r C =rr AM cA =G r APT Crr 182 186 192 Ala Cys lau Ser Leu Cys His Ile Glu Thr Glu Arg C MAM TM 1A C cAT T ciG AG (;AA AGA 31 Information for SEQ ID NO: 58 G6V SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser le Pro Pro Giu Asn His CIA ma c= A C PCA AAA Ck CZ aT AAT MA 7I Wk A: =M CM GMA NC 0U 160 173 176 GIn Asp His ?,sn Mn Ehe GIn Thr Lea Pro Tyr Val Pro Cys Ser Thr Cys Giu Gly Mn Lea CPA G;C CFC AC APC TIC CAA A CI aC TPT T *r Ar AA 1Cr GPA GT Are Crr 182 186 190 Ala Cys Leu Ser Iea Cys His Ile Glu Thr C a~ m= Tm 7cA CIC 7M C7' ATr GG APG 3' Information for SEQ ID NO: 59 G7V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Asn His Gin Asp His Asn Asn Pe Gin Thr Lea Pro Tyr Val Pro Cys Ser Thr Cys Ac= CPU' A GC CAC PC MC TIC CA P CIC OXVT GIT I 1m AGr PC 1 182 186 190 Glu Gly Asn Leu Ala Cys Leu Ser Leu Cys His Ile Glu Thr C GAA G3T AAT Cr Gzk T(M TIA CCIC= CAT G POG 3' Information for SEQ ID NO: 60 G200V5C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 61 amino acids, 183 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein WO 99/49892 -23 PCT/FR99/00703 140 N leA Pro Ala Thr Arg Lys Pro Pro lie Am Pro Ser Gly Ser Ile Pro Pro Giu Asn His cma aA a= Ac A AAA cA cA AiT An cm GPm aA n A mo aPG i c- G AAC CAT 160 173 176 GIn Asp His Asn Asn Ph Gin Thr Le Pro Tr Val Pro Ser Ser Th Cys Glu Gly Asn Le CA Gc w7 APc z CC CAA AM C'C VC G r P AGr ACA wr GAA a r APT Crr 182 186 200 Ala Cys La Ser Leu Ser His Lie Glu Tr G.u Mg Ala Pro Sex Arg Ala Pro Thr le C GM- 7W TIA7M CIC AC E r AT 1) AM GA AGA GA PM PGA GM CCO PEA -MC 3' Information for SEQ ID NO: 61 G198V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 59 amino acids, 177 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pr Pro Glu Asn His rA CCA GC AC PAM AAA CC AM AlT O CA TM G% P Mc 00k CM GPA XrC Cur 160 173 176 Gin Asp His Asn Asn Phe Gin thr Lea Pro Tyr Val Pro Ser Ser hr Cys Glu Gly Asn Leu CA cC C7C Ac AC TIC CAA A CZC TAT GIT PMZ PGr PA Mcr GPV GWr NC Crr 182 186 198 Ala Cys Leu Ser Leu Ser His Ile Glu Thr Glu Arg Ala Pro Ser Ang Ala Pro C GM X Ta TA C=C Xr C; AT G2FG AM GPA AMPGM\ CD\ PMI AGDM CM 3' Information for SEQ ID NO: 62 G196V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 57 amino acids, 171 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lea Pro Ala Th Arg Lys Pro Pro le Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His Ck a PG= ACA AG N OAA ATC PAT.CM 7M GA AM AM CM CM GA APC C7C 160 173 176 Gin Asp His Asn Asn FRh Gin 7rw Lea Pro Tyr Val Pro Ser Sex Thr Cys Glu Gly Asn LeA CM (7 C7C AAC APC TIC CAA P7 OX VC Gc r O PM AGT P CA GPA GT AV CIT 182 186 196 Ala Cys Leu Sex Lea Sex His le Giu Thr Glu Arg Ala Pro Ser Arg C G'kITG TrA ia d= A xr AT G G AaG @A iA am am Am Am 31 Information for SEQ ID NO: 63 G194V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 55 amino acids, 165 nucleotides STRANDEDNESS: single WO 99/49892 -24 PCT/FR99/00703 TOPOLOGY: linear MOLECULE TYPE: protein 140 N LAu Pro Ala Thr Arg Lys Pro Pro le Asn Pro Ser cly Ser nle Pro Pro Glu Asn His CI Ca GM ACA AGR h A OM ATT AI COM 7M A AM XC M G'A nAM CC 160 173 176 Gin Asp His Asn Asn Pie Gin Thr ieu Pro Tyr Val Pro Ser Se Ithr Cys Glu Gly Asn Leu CA CC- CAC APC ANC TIC C A Px CI= CIT a3= A=G AGr AMa r A~ CIT 182 186 194 Ala Cys Ieui Ser leu Ser His le Giu Thr Glu Arg Ala Pro C Ga Tnk 7M CIC A: CFX PZT OG ACG A AGA c pEG CC 3' Information for SEQ ID NO: 64 G192V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 53 amino acids, 159 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Leu Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His CIA Ca Go= ca AGA AM CCA CA ATT AX CM WA C M C AC C M G A C C 160 173 176 Gn Asp His Asn Asn Pte Gn Thx Lu Pro yr Val Pro Ser Ser Thr Cys Giu Gly Asn Leu CAA CGC cC AC AFC TIC CAA PC C Oc T GIT 1 P G PCh rmr GAA GM r ACM CIT 182 186 192 Ala Cys Lea Ser Leu Sex His Ile Glu Thr Glu Arg C GM 7W TM 7CA C C AM MlXT G;G ACG GMA 3' Information for SEQ ID NO: 65 G6V8C SEQUENCE LENGTH: 51 amino acids, 153 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 140 N Lau Pro Ala Thr Arg Lys Pro Pro Ile Asn Pro Ser Gly Sex Ile Pro Pro GI Asn His cmA Ca PCm PnA APA C cm AWT AAr Cm 7a PGsk AIMC i MC= @A Ap cCT 160 173 176 Gin Asp His Asn Asn Phe Gin Thr lea Pro Tyr Val Pro Sex Ser Mhr Cys Glu Gly Asn Le CAA GAC CC Abr A TIC CAA ACA C 0 IT AGC APACA TGT Gh WG AT CrI 182 186 190 Ala Cys Lea Sex Lea Ser His Ile Glu Itw C GM 7M 7CA C= A CC AT CG Xr 3' Information for SEQ ID NO: 66 G7V8C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 33 amino acids, 99 nucleotides WO 99/49892 25 PCT/FR99/00703 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein 158 173 176 N Asn His Gin Asp His Asn Asn Rp Gn Thr Leu Pro Tyr Val Pro Ser Ser Thr Cys AFC car A CC C C ATIC CPA AP cl aCX r GT =n A AGr aM r 182 186 190 Glu Gly Asn leu Ala Cys Laeu Ser Iaeu Ser His Ile Giu Thr C GAA GGT' AC CrrIT GA TI At CIC G CAT ATI Gr. 3' Information for SEQ ID NO: 67 G4V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Cys Ser Thr Cys GLu Gly Asn Leu Ala Cs 1eu Ser Leu Cys His C Grr r GC AGT Am =iT GA cr AAT CiT Ga T= Ti lA CIC IOC CAT 3' Information for SEQ ID NO: 68 G4V6C SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 17 amino acids, 51 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His C GIT COC AC PGr ACA GI' @A Gcr AA cTT =G 7W TIA TCLA CIT AC CAT 3' Information for SEQ ID NO: 69 G4'V SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Asp Ser Thr Asp Glu Gly Asn Leu Ala COm leu Ser Leu Orn His C Information for SEQ ID NO: 70 G4'V6C SEQUENCE TYPE: amino acids WO 99/49892 26 PCT/FR99/00703 SEQUENCE LENGTH: 17 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 171 173 176 182 186 187 N Val Pro Ser Ser Thr Asp Glu Gly Asn Leu Ala Orn leu Ser Leu Ser His C Information for SEQ ID NO: 71 G1V SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Ihr Cys GLu Gly Asn Leu Ala Cys Leu Ser Leu Cys His C PGr ACA mGT GhA Gr AT CIT Gm TGC TI TCA CIC 'GC CAT 3' Information for SEQ ID NO: 72 SEQUENCE TYPE: amino acids and nucleotides SEQUENCE LENGTH: 14 amino acids, 42 nucleotides STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser Thr Cys Glu Gly Asn Leu Ala Cys Leu Ser Leu Ser His C AGT ACA IT GAA GCT AAT CT GC TOC TIA A CIC AGC CT 3' Information for SEQ ID NO: 73 G1'V6C SEQUENCE TYPE: amino acids SEQUENCE LENGTH: 14 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N Ser hr Asp GLu Gly Asn Leu Ala Qmn Lu Ser Leu Ser His C Information for SEQ ID NO: 74 Gl' SEQUENCE TYPE: amino acids WO 99/49892 27 PCT/FR99/00703 SEQUENCE LENGTH: 15 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide 174 176 182 186 187 N er Ile APSr AsnAs PM W thr rp Ala Ile Ser Ls (ys C

Claims (12)

1. 05-JUN-2003 18:16 i P.12/14 Claims I. Use of at least one fragment of an enterobacteTium membrane protein OmpA for preparing a pharmaceutical composition adapted for nasal administration to improve the immunity of a mammal with respect to an antigen or to a hapten. 2. Use of at least one fragment of a membrane protein according to claim 1, characterized in that said enterobacteriumn membrane is a membrane of Klebsiella. 3. Use of at least one fragment of a membrane protein according to claim 2, characterized in that the membrane protein is an OmpA of Klebsiellapneumoniae. 4. Use of at least one fragment of a membrane protein according to one of clahims 1 to 3, characterised in that said membrane protein or its fragment is obtained by recombinant process. Use of at least one fragment of a membrane protein according to claim 4, characterized in that said recombinant membrane protein or its fragment is 15 renatured in the presence of detergent chosen from Zwittergent 3-14, Zwittergent 3-12 and octylglucopyranoside. Use of at least one fragment of a membrane protein according to one of claims 1 to 5, characterized in that at least one fragment has the sequence SEQ ID No 1.
2. 7. Use according to one of claims 1 to 6, characterized in that the antigen or the hapten are chosen from the group comprising proteins, peptides, So polysaccharides, oligosaccharides and nucleic acids. g S8. Use of at least one fragment of a membrane protein according to one of claims 1 to 7, characterized in that the antigen or the hapten originate from a virus 25 or from a bacterium. 090 9
3. 9. Use of at least one fragment of a membrane protein according to one of claims I to 8, characterized in that the antigen or the hapten comprises at least one protein fragment of a microorganism responsible for pathologies of the airways. COMS ID No: SMBI-00283893 Received by IP Australia: Time 18:15 Date 2003-06-05 05-JUN-2003 18:16 "7 P.13/14 Use according to claim 9, characterized in that said microorganism responsible for pathologies of the airways is chosen from RSV, parainfluenzat virus (PlM), influenza virus, hantavirus, streptococci, pnOumococCi and meningococci.
4. 11. Use of at least one fragment of a membrane protein according to one of claims 1 to 10, characterized in that the antigen or the hapten comprises at least one protein fragment of the human or bovine respiratory syncytial virus (RSV).
5. 12. Use according to claim 11, characterized in that the antigen or hapten comprises at least one fragment of the protein G of the RSV. t0
6. 13. Use according to either of claims 11 and 12, characterized in that the antigen or the hapten comprises at least one of the sequences SEQ ID No 2 to SEQ ID No 74.
7. 14. Use according to one of claims 1 to 13, characterized in that said fragment of a membrane protein is covalently coupled to said antigen or hapten. 15 15. Use according to claim 14, characterized in that one or more bonding elements is introduced into the fragment of membrane protein and/or of the antigen or of the hapten in order to facilitate the coupling.
8. 16. Use according to claim 15, characterized in that the bonding, element introduced is an amino acid.
9. 17. Use according to claim 14, characterized in that the hybrid protein, which is obtained after coupling between the fragment of a membrane protein and the antigen or the hapten, when said antigen or hapten is protein in nature, is prepared oo.. by genetic recombination,
10. 18. Use according to one of claims 14 to 17, characterized in that the i. 25 pharmaceutical composition contains a fragrent of a membrane protein coupled S* to an antigen or a hapten.
11. 19. Use according to claim 17, characterized in that the phanmaceuctical composition contains a transformed host cell which is capable of expressing a COMS ID No: SMBI-00283893 Received by iP Australia: Time 18:15 Date 2003-06-05 05-JUN-2003 18:16 P.14/14
12. 004308052. hybrid protein containing said fragment of membrane protein coupled to said antigen or hapten. Use according to either of claims 18 and 19, characterized in that the pharmaceutical composition does not contain any adjuvant. 21. Method for preparing a protein or one of its fragments by recombinant process, characterized in that said protein or one of its fragments is, after extraction, renatured in the presence of a solution comprising a detergent chosen from Zwittergent 3-14, Zwittergent 3-12 and octylglucopyranoside, and in that said recombinant protein is an enterobacterium membrane protein OmpA. 22. A use according to claim 1, substantially as described herein with reference to the Examples. 23. A method of improving the immunity of a mammal with respect to an antigen or to a hapten comprising nasally administering at least one fragment of an enterobacterium membrane protein OmpA to the mammal. 15 24. A method according to claim 23 substantially as described herein with S reference to the Examples. 25. A method according to claim 21 substantially as described herein with reference to the Examples. Dated this 5 th day of June 2003 ooo* Pierre Fabre Medicament 4 0 by its attorneys Freehills Carter Smith Beadle TOTAL P.14 COMS ID No: SMBI-00283893 Received by IP Australia: Time 18:15 Date 2003-06-05