AU733845B2 - Immunogenic complex, use, method of preparation thereof and vaccine containing same - Google Patents

Description

WO 97/41888 1 PCT/FR97/00800 IMMUNOGENIC COMPLEX, USE, METHOD OF PREPARATION THEREOF AND VACCINE CONTAINING SAME The present invention relates to new immunogenic complexes comprising a saccharide derivative, which are useful in particular as medicaments and in particular as vaccines.

Proteins and polysaccharides are the two main types of surface antigen encountered in bacteria and fungi and are, by virtue of their antigenic character, excellent tools which may be used in designing vaccines. However, the development of defined vaccines lacking side effects requires the use of vaccinating antigens of low molecular mass, mainly peptides or oligosaccharides. These antigens, but also others of higher molecular mass such as polysaccharides, cannot induce on their own an immune response which is intense and long-lasting.

The potential of bacterial polysaccharides in the preparation of vaccines appeared at the beginning of the XXth century. These compounds indeed play a major role in the structure and the pathogenicity of certain Gram-positive and -negative bacteria. Two types of bacterial polysaccharides are excellent candidates as vaccinal agents. They are the polysaccharides derived from the bacterial capsule and the polysaccharides derived from the lipopolysaccharides

(LPS)

of the outer membrane of Gram-negative bacteria.

They are polymers which are essentially composed of a carbohydrate portion, and in which a portion of the molecule is exposed at the surface of the bacterium. They consist of a linear chain of repeating units which are characteristic of a given bacterial species and of which the number may vary from one to several hundred, thus explaining their sometimes very high molecular weight. Each repeating unit itself consists of several monosaccharides linked to each other by glycoside bonds, generally from 1 to 7 monosaccharide residues. The latter may become substituted 2 to a greater or lesser degree with inorganic groups such as phosphates or with organic groups such as acids, amines, alcohols, fatty acids or amino acids.

The repetitive character of the epitopes of bacterial polysaccharides (small number of different epitopes), whether they are polysaccharides isolated from the bacterial capsule or lipopolysaccharides of the outer membrane of Gram-negative bacteria, indeed makes them T-independent immunogens. This results in the absence of an immune response to these antigens in young children and in the absence of an immune memory in adults (no cell-type immune response, little or no recall effect, limited antibody response to the IgM class).

Vaccines comprising a polysaccharide extract have proved relatively effective in adults at a low dose, 25 to 50 ig. Vaccines based on bacterial polysaccharides include to date vaccines against Neisseria meningitidis infections: a tetravalent vaccine against the group A, C, W135 and Y strains, Streptococcus pneumoniae infections: a multivalent pneumococcal vaccine combining 23 serotypes of capsular polysaccharide, Salmonella typhi infections: a vaccine composed of S. typhi capsular polysaccharide. However, these vaccines are particularly ineffective in young children.

Other approaches have been tested in the vaccine strategy against Salmonella infections. Indeed, bacteria of the genus Salmonella are virulent enterobacteria with digestive tropism, which are pathogens for humans and for numerous vertebrate animals. The genus Salmonella typically consists of more than 2000 different serotypes. The principal pathologies induced by these bacteria are (for general reviews see: Le Minor, L, 1987, Salmonella in Bact6riologie M6dicale, L. Le Minor and M. V4ron Eds., M6decine- Sciences Flammarion Paris, p. 411-427, Pegues, D.A. and 3 Miller, 1994, Salmonellosis including typhoid fever, Curr. Opin. Infect Dis. 7, 616-623): in animals, toxiinfections: specific to certain species, Salmonella abortus-ovis in ovines, Salmonella galinarum in poultry nonspecific, caused by the so-called "ubiquitous" serotypes (Salmonella Typhimurium, enteritidis, and the like), in humans: typhoid fevers (Salmonella typhi) and paratyphoid fevers (Salmonella paratyphi A, B and D), food toxiinfections, for which the serotypes most frequently blamed are Salmonella typhimurium, enteritidis and panama.

Currently, three types of anti-typhoid vaccine are available on the market (for general reviews see: Levine, Hone, Stocker, B.A.D. and Cadoz, 1990, New and improved vaccines against typhoid fever in New Generation Vaccines, G.C. Woodrow and M.M. Levine Eds, Marcel Dekker Inc. New York and Basel, p. 269-287, Jalla, Sazawal, S. and Bhan, 1994, Advances in vaccines for typhoid fever, Indian J. Pediatr. 61 321-329): The inactivated vaccines The injectable forms of the "inactivated bacterial vaccine" type are the oldest vaccine forms.

They are vaccines which may contain 500 to 1000 million bacteria per dose, in liquid form. The bacteria are inactivated by treatments with heat and/or with chemical compounds such as acetone, formalin or phenol.

Depending on the bacterial serotypes which they contain, there are the anti-typhoid vaccines: present in this class are the specialties "Typhoid vaccine" from the company WYETH-AYERST in the United States, or "Typhoid monovalent" from the company WELLCOME in the United Kingdom, the anti-typho/paratyphoid vaccines: present in this second class are the vaccines "Vac TAB" from 4 PASTEUR in France or "Typhidrall" from BIOCINE SCLAVO in Italy.

In France, the vaccine TAB (PASTEUR) is a trivalent liquid inactivated whole bacterial vaccine combining Salmonella typhi, Salmonella paratyphi A and B. Its relative efficacy is in fact limited to the T valency. This vaccine is reactogenic: it causes, in a third of cases, premature local and general reactions.

It is injected by the subcutaneous route at a rate of 2 to 3 injections, at 2 to 4 week intervals, followed by a booster. In France, the current regulations which still imposes it on some professional groups, including the military and health professions, are in fact no longer justified. In the military establishment, a noncommercialized monovalent T vaccine is now used.

The immunization of children under one year is generally not recommended because of the undesirable effects which may occur after the injection (considerable pain and high fever).

An attenuated live vaccine The oral typhoid vaccine Ty2la ("Vivotif", BERNA) contains a defective live strain of Salmonella typhi, lacking galactose-4-epimerase. It is administered orally, in the form of gastroresistant capsules containing 1 to 8 x 109 live bacteria in freeze-dried form. The vaccination scheme comprises 3 successive doses on days i, 3 and 5, with simultaneous doses of sodium bicarbonate to neutralize the gastric acidity.

Evaluated in endemic regions in Egypt and Chile, the protective value is thought to be between 60 and There are not thought to be any undesirable effects.

The vaccinal protection becomes effective approximately days after the last dose. The protection being effective for a period which may range from 1 to 7 years; it is thus recommended to those traveling to endemic regions to repeat the vaccination yearly.

Older oral forms are still available. They are inactivated vaccines whose efficacy has, however, not 5 been clearly established ("Taboral" from the company BERNA, "Enterovaccino" in Italy).

A subunit-type vaccine.

The vaccine "Typhim Vi" from the Institut MERIEUX is prepared from a capsular polysaccharide Vi purified from Salmonella typhi. It is an injectable solution containing 25 jig of polysaccharide. A single subcutaneous or intramuscular injection provides protection solely against the infectious risk linked to Salmonella typhi in adults and children over 5 years.

This vaccine does not give protection against Salmonella paratyphi A and B, these serotypes not being encapsulated. The immunity appears approximately days to 3 weeks after the injection. The duration of protection is equal to at least 3 years. In high endemic territories, the level of protection observed is about None of these specialties is sufficiently effective in the treatment and prevention of typhoid fever. The duration of protection is often limited. The vaccination of children is difficult and that of breast-feeding infants impossible. The inactivated vaccines can under no circumstances be prescribed for children and breast-feeding infants and the latter two vaccine forms cannot, for their part, be used in breast-feeding infants and young children. The oral vaccine Ty2la is indeed not recommended in children under 6 years and the polysaccharide vaccine "Typhim Vi" cannot be injected into children under 18 months.

In order to avoid these disadvantages, it would be desirable to be able to have immunogenic complexes capable of providing good immunity against the pathogenic strains, by causing both a humoral and cellular type immune response, in children and breastfeeding infants, the induction of a memory effect, and causing few side effects.

These aims, and others which will appear subsequently, can be achieved by means of immunogenic complexes, characterized in that they consist of at 6 least one oligo- or polysaccharide epitope naturally present in pathogenic agents such as bacteria, coupled to a carrier protein chosen from the human serum albumin binding protein of Streptococcus, the outer membrane proteins of a gram-negative bacterium, or fragments thereof. Indeed, the coupling, by a covalent bond, of the oligosaccharide or of the polysaccharide transforms them into T-dependent immunogens.

The oligo- or polysaccharide epitope is capable of being obtained from gram-negative or gram-positive bacteria, and in particular from membrane lipopolysaccharides or capsule oligosaccharides of bacteria of the genus Salmonella, Escherichia, Neisseria, Shigella, Haemophilus or Klebsiella.

According to a preferred aspect of the invention, the pathogenic agent may be Salmonella typhi, Haemophilus influenzae, Neisseria miningitidis or Streptococcus pneumoniae.

The oligo- or polysaccharide epitope may also be obtained from a fungus, in particular belonging to one of the genera Candida, Cryptococcus or Lipomyces.

The polysaccharides derived from lipopolysaccharides are prepared by extracting LPSs from the membrane and then removing lipid A by controlled hydrolysis. The capsular polysaccharides are, for their part, more easily isolated from the bacterial suspension: heating at 100 0 C for about ten minutes (solubilization) followed by centrifugation makes it possible to isolate the capsular polysaccharide Vi from Salmonella typhi. Both types of polysaccharides can then be purified by chromatography and/or membrane filtration.

The use of "whole" polysaccharides in a coupling method may give rise to certain technical problems due mainly to the excessively large size of these compounds: formation of a gel, precipitation.

ScTo overcome this problem, various methods of cleaving the polysaccharide may be used: fragmentation with ultrasound, depolymerization by oxidation- 7 reduction, controlled hydrolysis in an acidic or basic medium, enzymatic hydrolysis.

The method of fragmentation allowing the release of oligosaccharides should be appropriate for the polysaccharide studied. An oligosaccharide is a compound which may be derived from a polysaccharide and which has, compared with the initial polysaccharide, a reduced number of saccharide repeating units.

The Applicant has now demonstrated that the coupling, by a covalent bond, of the oligosaccharide or the polysaccharide with a carrier protein as defined above made it possible to solve the problems posed by the use of polysaccharide derivatives alone, or coupled to other carriers.

For example, the only conjugated vaccine on the market is a vaccine intended for the prevention of invasive Haemophilus influenzae type b infections (meningitis) in humans. For the four specialties commercialized, the conjugated vaccinating antigen is an oligosaccharide or a polysaccharide isolated from the capsule: PRP, polyribosyl ribitol phosphate.

The carrier proteins used in the design of this conjugated vaccine are of two types: the tetanus toxoids (TT) and diphtheria toxoids

(DT),

an extract of Neisseria meningitidis membrane proteins, OMPC.

The tetanus and diphtheria toxoids are currently the best characterized carrier proteins, both from a structural and biological point of view (vaccinal properties, carrier protein properties) and are, for these reasons, considered as the reference carrier proteins.

These proteins are used in the context of antitetanus and antidiphtheria vaccinations. The corresponding vaccines are very effective (protection close to 100% in both cases) and are well tolerated.

J- They are bacterial exotoxins which may be extracted and purified from a culture filtrate: 8 Clostridium tetani for TT and Corynebacterium diphtheria for DT. The TT toxin is a protein of 150 kDa. The DT toxin has a lower molecular mass and is secreted in the form of a single polypeptide chain of 535 amino acids. After purification, these proteins are inactivated by heat and formalin. They may be combined with each other (DT vaccine), and with many other vaccines (whooping cough, poliomyelitis and the like).

Being heat-resistant, they can be preserved at +4 0 C for a few years, but should not be frozen.

The too frequent use of these proteins (antitenus and antidiphtheria vaccination, conjugated vaccines) can however be incompatible with an intense response against the hapten. Their too frequent use has indeed the risk of causing the immune response to be aimed predominantly against these proteins, if the immunized subjects already have high levels of antibody against them.

The second type of carrier used in this same vaccine is in fact an extract of membrane proteins: OMPC, "Outer membrane protein complex", isolated from Neisseria meningitidis. This vesicular complex in fact contains several proteins combined with lipids and lipopolysaccharides.

According to a preferred aspect of the invention, the carrier protein comprises at least a portion of an OmpA-type protein of gram-negative bacteria, in particular a Klebsiella pneumoniae outer membrane protein.

Proteins which are particularly suitable for carrying out the present invention are proteins derived from the major membrane protein of Klebsiella pneumoniae 1-145, designated hereinafter p40; they have in particular one of the sequences ID No. 2, ID No. 4 or ID No. 6. Other carrier proteins of interest derived from the K. Pneumoniae outer membrane protein comprise fragments containing the third and fourth extramembrane loop flanking an intramembrane sequence, 9 containing an invariable extramembrane loop and the adjacent intramembrane sequence.

Invariable extramembrane loops are defined as the P40 sequences which are homologous with the sequences of the loops conserved between different species of enterobacteria. The sequences of the extramembrane loops which are not conserved during evolution are called variable loops. The location of the extramembrane loops is carried out according to the VOGEL and JAHNIG model (1986, J. Mol. Biol., 190: 191- 199) relating to the E. coli OmpA.

In particular, the fragments between amino acids 127 and 179 of sequence ID No. 1 will be used.

Other appropriate sequences are respectively the sequences between amino acids 108 and 179 of sequence ID No. i, amino acids 1 and 179 of sequence ID No. i, as well as sequences having at least homology with the preceding sequences.

According to another advantageous aspect of the invention, the carrier protein comprises all or part of the human serum alubumin binding domain of the streptococcus G protein (called hereinafter BB) This protein has a molecular mass of 29 kDa and may be expressed and produced in Escherichia coli in the form of inclusion bodies.

The carrier protein may in particular have the sequence ID No. 8, or a sequence having at least and preferably at least 90% similarity with said sequence ID No. 8.

All these carrier proteins may be extracted from source bacteria or alternatively may be obtained by the recombinant DNA route.

Immunogenic complexes according to the invention may in particular consist of a conjugate between a carrier protein as defined above and at least one substantially purified oligosaccharide, capable of being obtained from membrane lipopolysaccharides of bacteria of the genus Salmonella; in particular, the bacterium of the genus Salmonella will belong to a 10 serogroup carrying an antigenic specificity chosen from the following group: 0:1, 0:2, 0:4, 0:6, 7, 8, 0:3 and 0:9. In a preferred embodiment, the immunogenic complex contains an oligosaccharide capable of being obtained from the lipopolysaccharide of Salmonella enteritidis having the antigenic specificity 0:9.

Indeed, the various Salmonella serotypes are identified by their antigenic formula; they are classified into different groups, according to their antigenic specificity 0.

For example, Salmonella typhi belongs to group D, having the specificity 0:9, likewise S. enteritidis, S. panama and S. dublin.

Among the other major polysaccharide specificities, there may be mentioned: serogroup A, having the specificity 0:2, having as representative S. paratyphi A, serogroup B, having the specificity 0:4, having as representative S. paratyphi B and S. typhimurium, serogroup C, having the specificity 0:6, 7, 8, having as representative S. infantis and S. bovis morficans, serogroup E, having the specificity 0:3, with S. meleagridis.

The oligosaccharides belonging to the major antigenic specificities listed above will be particularly suitable for carrying out the invention.

For example, a vaccine is prepared from an oligosaccharide isolated from a lipopolysaccharide of S. enteritidis, carrying the antigenic specificity 0:9, will make it possible to protect against septicemias caused by Salmonella typhi and against typhoid fever, but it can also be used in the prevention, in humans and animals, of toxiinfections and zoonosis due to Salmonellae of the same serogroup.

Oligosaccharides according to one of the preferred aspects of the invention have at least one .unit: 11 aD galactose p(1-2)-aD mannose p(1-4)-aL Rhamnose p(1-3) atyvelose p(1-3) More particularly, the subject of the invention is an immunogenic complex comprising at least one oligosaccharide of formula a-Tyvp cD-Galp(l-2) aD-Manp(l-4) aL-Rhap(1-3) n in which Gal represents galactose Man represents mannose 10 Rha represents rhamnose Tyv represents tyvelose and n may vary between 1 and 24.

Preferably, n may vary between 1 and 5, and the oligosaccharide is coupled with a protein having one of the sequences ID No. 2, ID No. 4, ID No. 6 or ID No. 8, or possessing at least 80% similarity with one of the sequences ID No. 2, ID No. 4 or ID No. 6 or ID No. 8.

The subject of the invention is also the use of immunogenic complexes as defined above, for the 20 preparation of a vaccine; according to a particularly advantageous aspect, the complexes are useful for the preparation of a vaccine intended for protecting an animal against the infections caused by Salmonella bacteria belonging to the antigenic serogroup 0:9.

A mixture of oligosaccharides for which, in the above formula, n will have different values, may be used. The animal may be a human and is preferably a mamnal.

Immunogenic complexes according to the invention are also those comprising a Salmonella capsular antigen. The latter may be coupled alone to a Rn A carrier protein as defined above, or alternatively combined with a complex comprising another oligo- or 4, polysaccharide epitope.

12 The subject of the invention is also pharmaceutical compositions containing at least one oligosaccharide and/or antigenic complex as defined above. They may also contain other immunity adjuvants, and pharmaceutically acceptable excipients which are necessary for their formulation, such as a diluent, a stabilizer, preservatives and the like, known to persons skilled in the art.

According to an advantageous aspect, the invention relates to a vaccine containing a membrane oligosaccharide coupled to a carrier protein, and comprising, in addition, another antigenic determinant.

In particular, the vaccine comprises a Salmonella capsular antigen, such as the capsular antigen Vi (homopolymer of partially acetylated N-acetyl galacturonic); this makes it possible to enhance the efficacy of the vaccine against capsulated bacteria.

The method of preparing the immunogenic complex may comprise the following steps: a) Salmonella oligosaccharides are isolated from membrane lipopolysaccharides, b) optionally, the oligosaccharides are purified so as to keep oligosaccharides of the same molecular weight, c) the oligosaccharides are chemically activated, d) the activated oligosaccharides are coupled to a carrier protein in order to form the immunogenic complex.

According to a preferred aspect, the carrier protein is activated before step d) by a chemical method in order to facilitate the coupling.

The methods of activating the oligosaccharides with formation of an isothiocyanatophenylaminecontaining derivative and of coupling this derivative with a protein can be carried out as described by: McBroom et al. (1972, in: Methods in Enzymology, Vol. 28B, Ed. V. Ginsburg (Academic 4j/ Press, New York), pp. 212-219), or 13 Svenson S.B. and Lindberg A.A. (1979, J. Immunol.

Methods 25, 323-335).

Other methods may be used with the aim of activating the oligosaccharides and then coupling the derivatives obtained with a protein: methods involving sodium borohydride and cyanoborohydride, adipic acid dihydrazide, and the like.

This coupling may in particular follow the following scheme: 14 Oligosaccharide -OH H2N-{CH2)2- NH 2 NaCNBH3 Reductive amination Oligosaccharide -HN-(CH2)2- NH2 CsC2 Formation of an isothiocyanate Oligosaccharide -HN-(CH2)2-~ N=C=S HN Protein Coupling Oligosaccharide -HN-(CH2)2-( -NH-CS-NH Protein The coupling of oligosaccharides of different sizes may be envisaged. These oligosaccharides being liberated by enzymatic cleavage (endorhamnosidase activity of a phage), this will preferably consist of multiples of 4: tetrasaccharides, octasaccharides, dodecasaccharides, hexadecasaccharides,. icosasaccharides and the like. Likewise, the coupling of a mixture of these oligosaccharides (without prior purification) is included in the invention.

An additional step can then be carried out which consists of coupling the complex obtained at the end of step d) with another Salmonella antigenic determinant.

The subject of the invention is also the use of a protein comprising one of the sequences ID No. 2, 4, 15 6 or 8 for enhancing the immunogenicity of an oligosaccharide.

It also comprises the use of analogous proteins, in which at least one amino acid has been replaced with a homologous amino acid in the sequences ID No. 2, 4, 6 or 8.

The proteins will in particular be encoded by DNA sequences having one of the sequences ID No. 1, 3, or 7 or equivalent sequences, taking into account the degeneracy of the genetic code.

The sequence ID No. 2 represents the complete sequence of the protein It is also possible to use a recombinant protein designated LP40 (seq ID No. which comprises, in addition, a peptide of 9 amino acids comprising a portion of the leader sequence of the tryptophan operon.

Finally, the sequences ID Nos. 5 and 6 correspond to the entire transmembrane portion (AP40F8) and lack the highly immunogenic periplasmic portion (Puohiniemi, Karvonen, Vuopio-Varkila, J., Muotiala, Helander, I.M. and Sarvas, 1990, Infect. Immun. 58, 1691-1696).

The sequence ID No. 8 corresponds to the human serum albumin binding domain of the Streptococcus G protein.

The examples which follow are intended to illustrate the invention without limiting its scope in any manner.

In these examples, reference will be made to the following figures: Figure 1: Demonstration of the immunogenicity of the conjugate P40-icosasaccharide in mice.

Figure 2: Demonstration of the immunogenicity of the conjugate P40-icosasaccharide in rabbits.

16 EXAMPLE 1: Isolation and purification of the natural protein The P40 protein, a Klebsiella pneumoniae major outer membrane protein, is isolated by extraction in the presence of a detergent from the Klebsiella pneumoniae biomass, and then purified from the extract thus obtained by anion-, then cation-, exchange chromatography.

1.1. Materials and methods 1.1.1. Extraction of the membrane proteins The pH of the Klebsiella pneumoniae (strain 1-145, 40 to 340 g of dry cells, 7 to 10% of dry cells) is adjusted to pH 2.5 with pure acetic acid. After addition of 0.5 volume of a solution containing 6% cetrimide (detergent), 60% ethanol, 1.5 M CaC12 (final concentrations 2% cetrimide, 20% ethanol, 0.5 M CaC1 2 the pH is adjusted to 2.5 and the mixture is stirred for 16 hours at room temperature.

1.1.2. Clarification step After centrifugation for 20 min at 15,000 g at 4 0 C, the pellet is removed. The supernatant is an extract of Klebsiella pneumoniae membrane proteins.

1.1.3. Precipitation of the membrane proteins The proteins in the supernatant are precipitated by addition of 3 volumes of 95% ethanol at 0 C (final ethanol concentration After rapid stirring, the mixture is allowed to stand for 1 hour at 4 0 C minimum. The precipitated proteins are recovered by centrifugation for 10 min at 10,000 g at 4 0

C.

1.1.4. Preparation of a fraction enriched with membrane proteins (MP fraction) The pellets are resuspended in a 1% solution of Zwittergent 3-14 in an amount of 5 ml/g of wet pellet.

After stirring for 1 hour (propeller mixer) and grinding with the aid of an ultra-turrax (13,500 rpm, sec), the pH is adjusted to 6.5 with the aid of 1 N sodium hydroxide. Centrifugation of the mixture makes it possible to obtain the MP fraction (elimination of the insoluble matter).

17 1.1.5. Anion-exchange chromatography step The MP proteins are dialyzed overnight at 4 0

C

against a 20 mM Tris/HCl buffer pH 8.0, containing 0.1% Zwittergent 3-14. The dialyzate is deposited on a column containing a strong anion-exchange-type support (Biorad Macro Prep High Q gel) equilibrated in the buffer described above at a linear flow rate of cm/h. The proteins are detected at 280 nm. The protein is eluted, with a linear flow rate of 60 cm/h, for a concentration of 0.2 M NaC1 in the 20 mM Tris/HCl buffer pH 8.0 containing 0.1% Zwittergent 3-14.

1.1.6. Cation-exchange chromatography step The fractions containing the P40 protein are combined and concentrated by ultrafiltration with the aid of an Amicon stirred cell system used with a type Diaflo membrane (10 kDa cut-off) for volumes of the order of 100 ml, or with the aid of a Millipore Minitan tangential flow filtration system used with membrane plates having a 10 kDa cut-off for larger volumes. The fraction thus concentrated is dialyzed overnight at 40C against a 20 mM citrate buffer pH containing 0.1% Zwittergent 3-14. The dialyzate is deposited on a column containing a strong cationexchange-type support (Biorad Macro Prep High S gel) equilibrated in the 20 mM citrate buffer pH containing 0.1% Zwittergent 3-14. The P40 protein is eluted (rate 61 cm/h) for a 0.7 M NaCl concentration.

The fractions containing P40 are combined and concentrated as described above.

1.2. Results The fractions obtained after each chromatographic step are analyzed by SDS-PAGE in order to combine those containing the P40 protein.

After each step, the quantities of protein are determined by assay according to the Lowry method. The Spurity and homogeneity of the P40 protein are estimated by SDS-PAGE with stainings using coomassie blue and silver nitrate.

18 At the end of the purification process, P40 is concentrated with the aim of obtaining a protein concentration of the order of 5 to 10 mg/mL. The electrophoretic profiles reveal a degree of purity of greater than After immunoblotting, the protein is specifically recognized by an anti-P40 monoclonal antibody obtained in mice.

The presence of contaminating lipopolysaccharides (endotoxins) is estimated by the tube gelling method or LAL (Limulus Amebocyte Lysate) assay.

This assay which is carried out on the final solution reveals an endotoxin level of less than 160 EU/ml and therefore shows that said solution meets the standards of the European regulations.

EXAMPLE 2: Cloning, expression and purification of the recombinant P40 protein 2.1 Materials and methods 2.1.1. Cloning of the Klebsiella pneumoniae OmpA gene The nucleotide primers were determined from the published sequence portion of the OmpA of Klebsiella pneumoniae LD 199 (Lawrence J.G. et al., 1991, J. Gen.

Microbiol. 137, 1911-1921), the consensus sequence derived from the alignment of the OmpA sequences of different enterobacteria (Escherichia coli, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Enterobacter aeroginosae) as well as the peptide sequences obtained by sequencing, according to the Edman method, the natural protein isolated from Klebsiella pneumoniae 1-145 and the peptide sequences isolated after digestion with cyanogen bromide.

The oligonucleotides were synthesized according to the chemical phosphoramidite method with the aid of the Pharmacia Gene Assembler Plus apparatus.

A colony of Klebsiella pneumoniae 1-145 is lyzed in 10 p9 of lysis buffer (25 mM Taps pH 9.3, 2 mM MgC1 2 1 pi of this solution is then used as source of DNA for the PCR amplification reactions. These are 19 carried out in 100 il of amplification buffer with pmol of each primer and one enzymatic unit of Taq polymerase (Perclin Elmer Cetus). Each cycle comprises a denaturation step of 30 seconds at 950C followed by hybridization of the primer with the DNA and an extension of one minute at 720C. 30 cycles are thus carried out with the aid of a Perkin Elmer Cetus Gen Amp PCR 9000 thermocycler. The Klebsiella pneumoniae OmpA gene is cloned into the vector pRIT28 (Hultman T.

et al., 1988, Nucleosides Nucleotides 7, 629-638), a vector possessing the ampicillin resistance gene, the replication origins of Escherichia coli and of the Fl phage as well as a portion of the Escherichia coli Lac-Z gene (9-galactosidase).

The fragment thus cloned is sequenced with the aid of an Applied Biosystem automated sequencer 373 DNA Sequencer. The sequencing reactions are carried out with the aid of the dye terminator kit according to the supplier's recommendations.

2.1.2. Construction of the expression vector containing the Klebsiella pneumoniae OmpA gene The whole gene for the P40 protein is then cloned into the expression vector pTrp inducible by the presence of the tryptophan operon gene carrying the kanamycin resistance gene and having two BsmI and SalI restriction sites.

For the cloning, a BsmI restriction site is introduced by PCR upstream of the P40 gene, already having an SalI site downstream, into the vector pRIT28P40. The P40 gene having the BsmI/SalI sites is then cloned into the vector pTrp in order to constitute the plasmid 2.1.3. Expression of the LP40 protein The fusion protein LP40 is expressed and produced in Escherichia coli in the form of inclusion Sbodies. It will comprise the complete Klebsiella pneumoniae OmpA sequence to which there should be added at the level of the N-terminal end a peptide of 8 amino acids (peptide L) comprising a portion of the leader 20 sequence of the tryptophan operon necessary for the expression of the protein in Escherichia coli.

The expression of the LP40 protein is carried out in Escherichia coli RRIAM15 (Rtther, 1982, Nucl. Acid Res. 10 5765-5772). A preculture is carried out with stirring at 37 0 C overnight in a tryptic soybean broth basal medium (TSB medium) supplemented with yeast extract and in the presence of 30 g/ml of kanamycin. The operator region of the vector is blocked in the presence of an excess of tryptophan (100 gg/ml).

After reading the optical density at 580 nm, the culture is diluted in order to obtain an optical density of 1 in the preceding medium (TSB medium with yeast extract and kanamycin. The synthesis of the protein is induced by the addition of indoleacrylic acid (tryptophan analog) at the final concentration of ug/ml. The culture is kept at 37 0 C, with stirring for 5 hours.

2.1.4. Renaturation and purification of the protein After centrifugation (4000 rpm, 10 min, 4 0

C),

the cells are resuspended in a 25 mM Tris-HCl buffer pH Sonication allows the liberation of the inclusion bodies.

The pellet of inclusion bodies obtained by centrifugation (25 min at 10,000 g at 40C) is taken up in a 25 mM Tris-HCl buffer pH 8.5 containing 5 mM MgC12, and then centrifuged (15 min at 10,000 The denaturation of the protein is obtained by incubation of the inclusion bodies at 37 0 C for 2 hours in a 25 mM Tris-HCl buffer pH 8.5 containing 7 M guanidinium hydrochloride or urea (denaturation agent) and 10 mM dithiothreitol (reduction of the disulfide bridges).

Centrifugation (15 min at 10,000 g) makes it possible to eliminate the insoluble portion of the inclusion Sbodies.

After dilution with 13 volumes of a 25 mM Tris-HCl buffer pH 8.5 containing NaCl (8.76 g/l and Zwittergent 3-14 the mixture is left overnight at room 21 temperature with stirring in contact with air (renaturation by dilution and reoxidation of the disulfide bridges).

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 40C. The anion-exchange chromatography step is carried out on the Biorad Macro Prep High Q support as described above (Example The fractions containing the LP40 protein are combined and then concentrated by ultrafiltration before another dialysis against a 20 mM citrate buffer pH 3 containing 0.1% Zwittergent 3-14 (100 volumes of buffer) overnight at 40C. The cation-exchange chromatography step is carried out on the Biorad Macro Prep High S support as described in Example 1. The fractions containing are combined and then concentrated by ultrafiltration.

2.2. Results The Klebsiella pneumoniae OmpA gene comprises 1008 base pairs (sequence ID No. 1) and encodes a protein of 335 amino acids (sequence ID No.

2).

The LP40 gene comprises, for its part, 1035 base pairs (sequence ID No. 3) and encodes a protein of 344 amino acids (sequence ID No. As regards the OmpA gene portion, a few differences are observed at the level of the end encoding the two amino acids at the C-terminal position. These differences in fact relate only to three nucleotides. They are the nucleotides at position 1027 (C for G at position 1000 in the sequence of P40), 1028 (A for C at position 1001 in the sequence of P40), 1032 (C for T at position 1005 in the sequence of P40). These modifications are due to the use, during the cloning of the OmpA gene into the vector pRIT28, of a partially degenerate oligonucleotide primer (Kpnl4 sequence): A(G)G(C)CCTGCGGCTGAG3'. These modifications of the sequence of the gene cause a single difference between the peptide sequences of P40 and of LP40 which is 22 expressed and produced in Escherichia coli. It is the amino acid at position 343 of the sequence of which is a glutamine (Gln) residue whereas an alanine (Ala) residue is found in the sequence of (position 334).

Starting with 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 LP40 are purified after two chromatographic steps.

As above, the LP40 protein is concentrated after purification in order to obtain a final concentration of between 5 and 10 mg/ml. The electrophoretic profiles show a degree of purity of the order of 95%. After immunoblotting, the protein is specifically recognized by an anti-natural monoclonal antibody obtained in mice.

The state of the protein is monitored by SDS- PAGE. Depending on its form, denatured or native, the P40 protein extracted from the Klebsiella pneumoniae membrane has a characteristic electrophoretic (migration) behavior. The native form (0-sheet structure) indeed has a lower molecular mass than the denatured form (a helix structure) under the action of a denaturation agent, such as urea or guanidinium hydrochloride, or by heating at 100 0 C in the presence of SDS. The LP40 protein is not properly renatured after renaturation, whether the latter is carried out in the absence or in the presence of 0.1% (w/v) Zwittergent 3-14. On the other hand, complete renaturation is obtained after dialysis against a 25 mM Tris/HC1 buffer pH 8.5 containing 0.1% (w/v) Zwittergent 3-14. However, it should be noted that this renaturation is obtained only 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).

23 EXAMPLE 3: Cloning, expression and purification of the transmembrane portion of the P40 protein In order to clone the portion of the gene corresponding to the entire transmembrane portion lacking the periplasmic portion of the P40 protein, an oligonucleotide complementary to the end encoding the C-terminal portion of this region of the gene, a sequence between amino acids 1 and 179 of the protein and called fragment F8, was synthesized.

The sequence of the gene corresponding to the desired fragment was amplified by PCR from the DNA of a miniprep of the vector pRIT28P40, and then purified and cloned into the same vector. Sequencing was carried out in order to check that no mutation occurred during the amplification.

This gene was then cloned as described above (Example 2) into the vector pTrp in order to constitute the plasmid pTrpLAP40F8.

The fusion protein LAP40F8 is expressed in Escherichia coli RRI after transfection with the vector pTrpLAP40F8. After one denaturation/renaturation cycle, it is purified by anion- followed by cation-exchange chromatography as described above (Example 1).

The gene for the protein LAP40F8 comprises 567 base pairs (sequence ID No. 5) and encodes a protein of 118 amino acids (sequence ID No. 6).

EXAMPLE 4: Expression and purification of the BB protein 4.1. Materials and methods 4.1.1. Expression of the BB protein The gene for the BB protein is cloned into the expression vector pva inducible by the presence of the tryptophan operon gene, carrying the ampicillin and tetracycline resistant genes and possessing a replication origin in Escherichia coli. The BB protein is expressed and produced in Escherichia coli RV 308 (strain ATCC 31608) in the form of inclusion bodies.

24 The competent Escherichia coli RV 308 strains are transformed with the vector pvaBB. A preculture is carried out, with stirring, at 37 0 C overnight in a tryptic soybean broth basal medium (TSB medium) supplemented with yeast extract and in the presence of tetracycline (8 pg/ml) and ampicillin (200 gg/ml). The operator region of the vector is blocked in the presence of an excess of tryptophan (100 ig/ml).

After reading the optical density at 580 nm, the culture is diluted in order to obtain an optical density of 1 in the preceding medium (TSB medium with yeast extract and tetracycline/ampicillin). The synthesis of the BB protein is induced by the addition of indoleacrylic acid (tryptophan analog) at the final concentration of 25 ig/ml. The culture is kept at 37 0

C,

with stirring, for 5 hours.

4.1.2. Renaturation and purification of the BB protein After centrifugation (4000 rpm, 10 min, 4 0

C),

the cells are resuspended in a 25 mM Tris-HCl buffer pH 8.5. Sonication allows the liberation 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-HC1 buffer pH 8.5 containing 5 mM MgC12, and then centrifuged (15 min at 10,000 The denaturation of the protein is obtained by incubation of the inclusion bodies at 37°C for 2 hours in a 25 mM Tris-HCl buffer pH 8.5 containing 7 M guanidinium hydrochloride (denaturation agent) and 10 mM dithiothreitol (reduction of the disulfide bridges).

Centrifugation (15 min at 10,000 g) makes it possible to eliminate the insoluble portion of the inclusion bodies.

After dilution with 13 volumes of a 25 mM Tris- HC1 buffer pH 8.5 containing NaC1 (8.76 g/l and Zwittergent 3-14 the mixture is left overnight at room temperature with stirring in contact with air (renaturation by dilution and reoxidation of the disulfide bridges).

25 After another centrifugation, the BB protein is purified by affinity chromatography on an HSA-Sepharose support (support prepared by coupling human serum albumin to a "CNBr-activated Sepharose 4B" Pharmacia gel). After injection (low flow rate), the unbound proteins are eluted with a 25 mM Tris/HCl buffer pH containing 0.2 M NaC1, 0.05% Tween 20 and 1 mM EDTA.

The BB protein retained on the support is eluted with a M acetic acid solution pH 2.7. The fractions containing the protein of interest are combined and then concentrated by ultrafiltration.

4.2. Results The gene for the BB protein comprises 774 base pairs (sequence ID No. 7) and encodes a protein of 257 amino acids (sequence ID No. 8).

The protein expressed comprises, from the N-terminal end (see sequence ID No. 7): the peptide L, amino acids 1 to 8, the peptide amino acids 9 to 14, a linker peptide, amino acids 15 to 23, the BB protein, amino acids 24 to 257.

The protein produced has a molecular mass of about 29 kDa (analysis by SDS-PAGE).

EXAMPLE 5: Isolation and purification of the Salmonella enteritidis oligosaccharides from lipopolysaccharides 4.1. Preparation of the lipopolysaccharides (LPS) The Salmonella enteritidis SH 1262 bacteria are cultured in a 10-liter fermenter at 37 0 C, at pH with vigorous stirring, in a Ty medium. The cells are killed by addition of 1% formaldehyde and are recovered by centrifugation at 4000 g for 20 min at 4 0 C. After washing in PBS and another centrifugation, carried out as above, the pellet is resuspended at a concentration of the order of 20 mg (dry weight)/ml. The LPSs are extracted by the phenol method (Westphal O., LIderitz 0. and Bister 1952, Z. Naturforsch. 7, S 148-155) and the aqueous phase is recovered and freezedried.

26 LPSs which have been made partially lipid-free are prepared by hydrolysis of the phosphate and ester bonds at the level of the lipid portion (lipid A) by a treatment carried out in the presence of 0.15 M sodium hydroxide at 100 0 C for 2 hours. After centrifugation, the pH is adjusted to 3.5 and the free fatty acids are removed by successive extractions with chloroform. The pH is then adjusted to 7.0 before the LPSs which have been made lipid-free are dialysed against water and finally freeze-dried.

4.2. Preparation of the oligosaccharides The oligosaccharides are prepared from LPSs which have been made partially lipid-free by using the endorhamnosidase activity associated with the bacteriophage P36.

The LPSs obtained above are added to a dialysis tubing containing the P36 phage, dialysed beforehand against a 5 mM ammonium carbonate buffer pH 7.1 in a ratio of 1 g of LPS/10 14 p.f.u. of phage. The dialysis is carried out at 37 0 C against 600 to 800 ml of the preceding buffer. After 50 hours, the dialysis bath is changed and the dialysis is repeated for an additional period of about 40 hours. The two counter-dialysis solutions are then mixed and then concentrated with the aid of a rotary evaporator.

The oligosaccharides are fractionated by molecular sieve chromatography. The concentrated oligosaccharides are deposited on a Biogel P2 or P4 (Biorad, 200-400 mesh) column (2.5 x 170 cm) eluted with water (flow rate 8.5 ml/h). The fractions containing the oligosaccharides are detected by the phenol sulfuric acid) method. After analyzing the fractions by thin-layer chromatography, the fractions containing the different isomers are combined and then freeze-dried. The purity of the oligosaccharides obtained is determined by nuclear magnetic resonance $nd mass spectrometry.

27 EXAMPLE 6: Coupling of the oligosaccharides isolated from Salmonella enteritidis lipopolysaccharides to the protein The oligosaccharides (10 to 40 mg) are dissolved in 0.5 ml of water. This solution of oligosaccharides is added dropwise, with stirring, to 1 ml of a solution of para-aminophenylethylamine diluted with water containing 20 mg of sodium cyanoborohydride. After adjusting the pH to 8.0 with 1 N NaOH, the reaction mixture is left at room temperature for 24 hours. The excess of reagents is removed by gel filtration on a Biogel P2 column. The fractions containing the derived oligosaccharides are recovered, concentrated to dryness with the aid of a rotary evaporator and then the oligosaccharides are taken up in 3 ml of 80% ethanol.

The addition of 200 il of a solution of thiophosgene diluted in 80% ethanol (lv/3v) allows the production of isothiocyanato-p-aminophenylethylaminooligosaccharide derivatives. The pH is maintained with the aid of a 1 M sodium hydroxide solution in ethanol. After 2 hours at room temperature, the reaction is complete, and the derived oligosaccharides are separated from the excess of reagents by chloroform extraction (elimination of the thiophosgene). The aqueous phase is concentrated to dryness and the oligosaccharides are taken up in 0.5 ml of 0.1 M bicarbonate buffer pH 8.2 containing 0.1% Zwittergent 3-14.

The P40 (LP40 or AP40F8) protein dissolved in a 0.1 M bicarbonate buffer pH 8.2 containing 0.1% Zwittergent 3-14 (2.3 ml, concentration of the order of mg/ml) is added dropwise, with constant stirring, to the oligosaccharides. The pH is adjusted to 8.7 with 1 M sodium hydroxide and the reaction mixture is kept at room temperature for 48 hours. The noncoupled oligosaccharides are eliminated by a series of several dilutions and concentrations with the aid of an Amicon 4 stirred cell equipped with a Diaflo membrane having a 28 cut-off of 30 kDa. The conjugates obtained are dialyzed several times against 1 liter of PBS buffer containing 0.1% Zwittergent 3-14. The level of substitution is determined after estimation of the quantities of oligosaccharides by the assay method using phenol sulfuric acid), and of protein by the Lowry method. The conjugates are stored at -200C.

In the case of the conjugate icosasaccharide, the degree of substitution, estimated by assay of the proteins and of the oligosaccharides, is 5.3 moles of icosasaccharides/mole of P40. This value is in agreement with that established after SDS- PAGE of the conjugate.

EXAMPLE 7: Demonstration of the immunogenicity of the conjugates in mice 6.1. Materials and methods The mice (NMRI females, 1S20 g, 6/batch) are immunized on days 0, 14 and 21. Each mouse receives a dose of 0.5 ml of the conjugate prepared in Example 3.

The conjugates (10 pg) are injected in the presence or in the absence of complete Freud's adjuvant and the injections are carried out by the intraperitoneal route. Blood samples are collected by puncture on days 0 and 35. The antibody responses are evaluated on the individual sera collected by the ELISA method. The anti-icosasaccharide IgGs of the sera are isolated on a BSA-hexadecasaccharide support and are revealed with the aid of an anti-mouse IgG antiserum labeled with alkaline phosphatase. The optical density is determined at 405 nm.

6.2. Results When it is injected in the presence of complete Freud's adjuvant, the P40/icosasaccharide conjugate allows the induction of a high response directed against the oligosaccharide from day 35 (3 immunizations): the antibody titer is close to 1/1x10 5 As shown in Figure 1, this titer is maintained when the immunizations are carried out in the absence 29 of adjuvant. The anti-oligosaccharide antibody titers are indeed between 1/1x10 4 and 1/1x10 EXAMPLE 8: Demonstration of the immunogenicity of the P40/icosasaccharide conjugates in rabbits 7.1. Materials and methods The rabbits (New Zealand white rabbits, 2-3 kg) are immunized on days 0, 14 and 28. The oligosaccharide conjugates (10 gg) are injected into the animals into the popliteal lymph nodes in the presence or in the absence of complete Freud's adjuvant. On days 0, 14, 28 and 56, blood samples are collected and the antibody responses are evaluated on the individual sera by the ELISA method. The titer plates are coated with 2 different antigens: the protein and the LPS from which the oligosaccharides coupled to P40 are derived. The antibodies are revealed with the aid of an anti-rabbit IgG conjugate labeled with alkaline phosphatase. The optical density is determined at 405 nm.

7.2. Results The icosasaccharide, when it is presented by the P40 protein, induces, in the presence of Freud's adjuvant, a high response against the lipopolysaccharide from which it is derived: titer greater than 1/1x10 6 In the absence of adjuvant, the response directed against the icosasaccharide, when it is presented by P40, is lower than that obtained in the presence of adjuvant, but significantly higher than that induced after injection of the BSA-octasaccharide conjugate. Figure 2 presents the results of the ELISA assay carried out against the LPS from which the icosasaccharide coupled to the P40 protein is derived.

After 56 days, the antibody titer is greater than 1/10,000.

30 EXAMPLE 9: Challenge experiment in mice after passive transfer 8.1. Materials and methods The mice (NMRI females, 20 g, 6/batch) receive one injection, by the intravenous route, of 0.2 ml of a hyperimmune serum obtained in rabbits after one immunization cycle carried out under the conditions described above in Example 5 (injections in the absence of adjuvant, collection of serum on D56).

The challenge with Salmonella enteritidis SH 2204 bacteria is carried out 2 to 3 hours after injection of the hyperimmune serum. The bacteria are injected into the animal by the intraperitoneal route.

Three different doses are used: 1.3 13 and 52 times the LD50 (LD50 2.6x10 5 cells/ml). The mice are .observed for up to 60 days after the injection.

8.2. Results The antibodies obtained in rabbits after immunization with the P40-icosasaccharide conjugate make it possible, after injection by the intravenous route, to protect mice against a Salmonella enteritidis infection. 60 days after challenges carried out by injection of doses of the order of 1.3 and 13 times the all the mice were alive (Table the 52 x dose being, for its part, excessive (death of the animals).

Table 1: Challenge experiments in mice after passive transfer of a rabbit hyperimmune serum or after immunization with the P40-icosasaccharide conjugate.

Determination of the percentage of animals alive days after injection by the intraperitoneal route of a dose of Salmonella enteritidis bacteria.

Dose of Salmonella Challenge after Challenge after enteritidis passive transfer immunization 1.3 x LD50 100% 100% 13 x LD50 100% 100% 52 x LD50 O 0 31 EXAMPLE 10: Challenge experiment in mice after immunization with the P40/icosasaccharide conjugate 9.1. Materials and methods The mice (NMRI females, 20 g, 6/batch) are immunized with the P40/icosasaccharide conjugate in the absence of adjuvant as described in Example 4.

On day 42, the challenge with Salmonella enteritidis SH 2204 bacteria is carried out as described above in Example 6.

9.2 Results The vaccination with the conjugate makes it possible to directly protect the mice against a challenge with Salmonella enteritidis.

The immunizations with this conjugate indeed make it possible to increase the LD50 by a factor of 10 at the least (Table it being possible for the LD50 in this case to be greater than 3.4 x 10 6 /ml.

LEGEND TO THE FIGURES Figure 1:

DO..

Figure 2: 32

DO

-D14 D2S D56 It will be understood that the term "comprises" or its grammatical variants as used in this specification and claims is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

*o• *oo *o ooo o *oo ooo 33 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: PIERRE FABRE MEDICAMENT STREET: 45 PLACE ABEL GANCE CITY: BOULOGNE COUNTRY: FRANCE POSTAL CODE: 92100 (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: Apple Macintosh OPERATING SYSTEM: MAC OS Systeme 7 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: Length: 1008 base pairs Type: nucleic acid Strandedness: single Topology: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1008 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCT CCG AAA GAT AAC ACC TGG TAT GCA GGT GGT AAA CTG GGT TGG TCC 48 Ala Pro Lys Asp Asn Thr Trp Tyr Ala Gly Gly Lys Leu Gly Trp Ser 1 5 10 34 CAG TAT CAC Gin Tyr His GGT CCG ACC Gly Pro Thr GAC ACC Asp Thr GGT TTC TAC GGT AAC GGT TTC Gly Phe Tyr Gly Asn Gly Phe .25 CGT AAC GAT Arg Asn Asp CAG MT GGT GCT GGT GCG Gin Leu 40 Gly Ala Gly Ala CAG AAC AAC AAC Gin Asn Asn Asn TTC GGT GGT TAC Phe Gly Gly Tyr GAC TCC CTC GGC Asp Trp Leu Gly UTC AAA GCT CAG Phe Lys Ala Gin CAG GTT Gin Vol AAC CCG TAC CTC Asn Pro Tyr Leu TTC GMA ATG GGT Phe Glu Met Gly

CGT

Arg ATG GCA TAT AAA Met Ala Tyr Lys AGC CTT GAC AAC Ser Vol Asp Asn GCT GCT Gly Ala GGCC CUT CAG CTG Gly Vol Gin Leu GAC ATC TAC ACC Asp Ilie Tyr Thr 100 GCT AAA CTG GGT Ala Lys Leu Gly CCG ATC ACT GAC Pro le Thr Asp CAT CTG Asp Leu TCC AAA Ser Lys CGT CTG GGC Arg Leu Gly GGC ATG Gly Met 105 GT T TCC Vol 5cr 120 GTT TGG CGC GCT Val Trp Arg Ala GGC AAC TAC Gly Asn Tyr 115 GCT TT ACC GGC Ala Ser Thr Gly CGT AGC Arg Ser GAC ACT GGC Asp Thr Gly CiT TCC Val Ser 130 CCA GTA Tr GCT Pro Val Phe Ala GGC GGC GTA GAG TGG GCT Gly Gly Val Giu Trp Ala 135 -144 CUT ACT CCT CAC Val Thr Arg Asp GCT ACC CGT CT GCAA TAC CAG TGG Ala Thr Arg Leu Glu Tyr Gln Trp 150 GTT AAC AAC Val Asn Asn 155 ATC CGC GAC le Gly Asp CCC ACT GTC GGT Gly Thr Val Gly CGT CCT CAT AAC GGC ATG CTG AGC CTG Arg Pro Asp Asn Gly Met Leu Ser Leu 170 GGC CU Gly Vol 175 TCC TAC CGC Ser Tyr Arg GGT CAG GAA CAT Cly Gin Glu'Asp GCA CCG CUT GUT Ala Pro Val Val GCT CCG GCT Ala Pro Ala 190 CCG CCT CCC Pro Ala Pro 195 GCT CCC CAA GTG Ala Pro Clu Val ACC AAG CAC TTC ACC CTG AAC TCT Thr Lys His Phe Thr Leu Lys Ser 35 GAC GTT Asp Val zie CTG TTC AAC TTC Leu Phe Asn Phe AMA GCT ACC CTG Lys Ala Thr Leu AAA CCG Lys Pro 220 GAA GGT CAG Glu Gly Gin (AG GCT CTG GAT CAG CTG TAC ACT CAG CTG Gin Ala Leu.Asp- Gln Leu Tyr Thr Gin Leu AAC ATG GAT CCG Asn Met Asp Pro

GAC

Asp GGT TCC GCT Gly Ser Ala GTT GTT Val Val 245 CTG GGC TAC ACC GAC Leu Gly Tyr Thr Asp 250 CGC ATC GGT Arg Ile Gly TCC GMA Ser Glu 255 GCT TAC AAC CAG .Ala Tyr Asn Gin 260 CAG CTG TCT 'GAG AAA CGT GCT (AG TCC GT MT GAC Gin Leu Ser Glu Lys Arg Ala Gin Ser Vol Vol Asp 26S 270 TAC CTG M1 Tyr Leu Vol 275 ATG GGT GAA Met Gly Glu 290 GCT AAA GGC Ala Lys Giy TCC AAC CCG 5cr Asn Pro ATC CCG Ile Pro 280 MT ACT Vol Thr 295 GCT GGC AAA ATC Ala Gly Lys Ile GCT CGC GGC Ala Arg Gly GGC AAC ACC Gly Asn Thr GAC AAC GTG AAA Asp Asn Val Lys CGC G&T GCC CTG Arg Aia Ala Leu GAT TGC CTG GCT CCG GAT CGT CGT GTA Asp Cys Leu Ala Pro Asp Arg Arg Vol 315 ATC GAA GTT AAA Ile Glu Vol Lys TAC AAA GAA Mi GTA ACT Tyr Lys Glu Val Val Thr 330 (AG CCG GCG Gin Pro Ala GGT TAA Gly 335 1008 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: Length 335 amino acids Type amino acid Strandedness :single Topology linear (ii) MOLECULE TYPE protein 36 (xi) SEQUENCE DESCRIPTION SEQ ID NO: 2: Ala Pro Lys Asp Asn Thr Trp Tyr Ala His Asp Asn Asp Gly Phe .Asp Asn Pro Ile Ala Asp 110 Thr Gly Ile Ala 145 Vol Gly Gly Gin Val Ala zoo Ala Thr Ser Asn 235 Ile Gly Vol Asp Phe Tyr Gly Ala Gly Tyr 60 Phe Lys Asp Leu Gly Asn Pro Val Leu Glu 150 Pro Asp Ala Ala His Phe Pro Glu Pro Lys .240 Ala Tyr Val Ala 275 Gly Phe Gi y Gly Vol Thr Thr Gly Vol Leu Ala 190 Se r Ala Ala Leu Pro 280 Gly Lys Leu Gly Trp Gln Asn Asn Asn Gly 30 Gly Tyr Gln Vol Asn Arg Met Ala Tyr Lys 65 Gin Leu Thr Ala Lys Arg Leu Gly Gly Met 105 Gly Val Ser Arg Ser 120 VIal Giu Trp Ala Vol 140 hsn Asn Ile Gly Asp 155 Ser Leu Gly Vol Ser 175 Pro Ala Pro Ala Pro 195 ksp Vol Leu Phe Asn 210 Leu Asp Gin Leu Tyr 230 Val Val Leu Gly Tyr Z45 Ser Glu Lys Arg Ala 26S kla Gly Lys Ie Ser Sea- Gin Tyr Pro Thr Arg 3S Pro Tyr Leu Gly Ser Vol Leu Gly Tyr Val Trp Arg Glu His Asp 125 Thr Arg Asp Ala Gly Tha- 160 Tyr Arg Phe 180 Ala Pro Glu Phe Asn Lys 215 Thr Gin Leu Thr Asp Arg 250 Gin Ser Vol 270 Ala Arg Gly 37 Met Gly Giu Ser Asn Pro Val Thr Gly Asn Thr Cys Asp Asn Vol Lys Ala Arg 290 295 300 305 Ala Ala Leu Ile Asp Cys Leu Ala Pro Asp Arg Arg Val Glu 310 315 320 Gly Tyr Lyj Glu Val Val Thr Gin Pro Ala Gly 325 330 335 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: Length: 1035 base pairs Type: nucleic acid Strandedness: single Topology: linear (ii) MOLECULE TYPE: DNA Ile Glu Vol Lys (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1035 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG AAA GCA AUT Met Lys Ala Ile 1 TTC GTA CTC AAT -CC GCT CCG AAA GAT Phe Val Leu Asn Ala Ala Pro Lys Asp 5 AAA CTG CGT TGG TCC CAG TAT CAC GAC Lys Leu Gly Trp Set Gin Tyr His Asp

AAC

Asn TAT GCA GGT Tyr Ala Gly TAC GGT AAC Tyr Gly Asn CTT GGT GCC Leu Gly Ala ACC GGT TTC Thr Gly Phe AAC CAT CAG Asn Asp Gin 96 GGT TTC CAG AAC MAC AAC GGT CCC ACC Cly Phe Gin Asn Asn Asn Gly Pro Thr GGT GCG TTC Cly Ala Phe GGT'GGT TAC CAG GTT MAC Cly Cly Tyr Gin Vol Asn 55 CCG TAC CTC GGT Pro Tyr Leu Cly CAA ATG GCT TAT Glu Met Gly Tyr TCC CTC CCC CGT Trp Leu Gly Arg GCA TAT AAA GCC Ala Tyr Lys Gly GTT GAC AAC GCT GCT TTC AAA Vol Asp Asn Gly Ala Phe Lys CTG GGT TAC CCG ATC ACT GAC Leu Cly Tyr Pro Ile Thr Asp GGC ATG GIT TGG CGC GCT GAC Gly Met Vol Trp Arg Ala Asp 115 GTT TCC CGT AGC GAA CAC GAC Vol Ser Arg 5cr Glu His Asp 130 135 GGC GTA GAG TGG GCT GTT ACT Gly Vol Glu Trp Ala Val Thr 145

ISO

CAG TGG GTT AAC AAC ATC GGC Gin Trp Vol Asn Asn Ile Gly 165 GAT AAC GGC ATG CTG AGC CTC Asp Asn Gty Met Leu Ser Leu 180 GAT CCT CCA CCC GTT GTT GCT Asp Ala A la Pro Vol Vol Ala 195 GCT ACC AAG CAC TTC ACC CTG Ala Thr Lys His Phe Thr Leu 210 215 AAA GCT ACC CTG AAA CCG GAA Lys Ala Thr Leu Lys Pro Glu 225 230 GCT CAG Ala Gin CAT CTC Asp Leu 105 TCC AAA 5cr Lys 120 ACT GCC Thr Gly CGT CAC Arg Asp CAC C Asp Ala CCC GTT Cly Vol 185 C GCT Pro Ala zoo AAG TCT Lys 5cr GGT CAG Gly Gin 38 CCC GTT Cly Vol 90 GAC. ATC Asp Ile CCC AAC Gly Asn GTU TCC Vol Ser ATC CCT Ile Ala 155 CCC ACT Gly Thr 170 TCC TAC 5cr Tyr CCC GCT Pro Ala CAC GTT Asp Vol CAG GCT Gin Ala 23S CTG ACC GCT AAA Leu Thr Ala Lys ACC CCT CTC GCC Thr Arg Leu Cly 110 CCT TCT ACC GCC Ala 5cr Thr Cly 125 CTA TTT GCT GCC Vol Phe Ala Gly CGT CTC CAA TAC Arg Leu Giu Tyr 160 CGT ACC CGT CCT Gly Thr Arg Pro 175 TTC GCT CAC CAA Phe Gly Gin Ciu 190 GCT CCG CAA GTG Ala Pro Giu Vol 205 TTC AAC TTC AAC Phe Asn Phe Asn CAT CAG CTG TAC Asp Gin Lcu Tyr 240 GCT CiT GTT CTG Ala Vol Vol Lcu 255 288 336 384 432 480 528 ACT CAC CTG Thr Gin Leu GCC TAC ACC Cly Tyr Thr ACC AAC ATG GAT CCG 5cr Asn Met Asp Pro AAA GAC GGT TCC Lys Asp Cly 5cr Z5@ GAA GCT TAC AAC Glu Ala Tyr Asn 265 CCC ATC CGT TCC Arg Ile Cly 5cr CAG CTG Gin Leu 270 CGT GCT Arg Alp GGC A Gly Lys AAC ACC Asn Thr GCT CCC Ala Pro TCC GTT CT Ser Vol Val 280 TCC GCT CC Ser Ala Arg 295 CAC AAC CTG Asp Asn Val 310O CGT CGT GTA Arg Arg Val 39- GAC TAC CTC Grr GCT AAA GCC Asp Tyr Leu Val Alo Lys Cly 285 GGC ATG GGT CAA TCC AAC CCC Gly Met Gly Glu Ser Asn Pro 300 AAA GCT CGC CCT GCC CTG ATC Lys Ala Arg Ala Ala Leu Ile 315 GAG ATC GMA GTT AAA GGC TAC Glu le Glu Vol Lys Gly Tyr 330 335 1008 103S GAA GTT GTA ACT Glu Vol Vol Thr 340 CCC CAG GCC TAA Pr-o Gin Gly INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: Length: 344 amino acids Type: amino acid Strandedness: single Topology: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ala Ile Phe Vol Leu Asn Ala Ala Pro iys Asp 1 5 10 Gly Gly Lys Leu Gly Trp, Ser Gin Tyr His Asp Thr Gly 25 30 Phe Gin Asn Asn Asn Gly Pro Thr Arg Asn Asp Gln Leu 45 Gly Gly Tyr Gin Vol Asn Pro Tyr Leu Gly Phe Glu Met 60 65 NO: 4 Asn Thr Phe Tyr Gly Ala so Gly Tyr Trp Tyr Ala Gly Asn Gly Gly Ala Phe Asp Trp Leu 40 Gly Arg Met Ala Tyr Lys Gly Ser Vol Asp Asn Gly Ala Phe Lys Ala Gin Gly 80 85 Vol Gin Leu Thr Ala Lys Leu Gly Tyr Pro le Thr Asp Asp Leu Asp le Tyr 100 105 Thr Arg Leu. Gly Gly Met Vol Trp Arg Ala Asp Ser Lys Gly Asn Tyr Ala Ser 110 115 120 125 Thr Gly Vol Ser Arg Ser Glu His Asp Thr Gly Vol 5cr Pro Vol Phe Ala Gly 1.36 13S 140 Gly Val Giu Trp Ala Vol Thr Arg Asp Ile Ala Thr Arg Leu Glu Tyr Gin Trp 145 150 155 160 Vol Asn Asn Ile Gly Asp Ala Gly Thr Val Gly Thr Arg Pro Asp Asn Gly Met 165 170 175 180 Leu Ser Leu Gly Vol 5cr Tyr Arg Phe Gly Gin Glu Asp Ala Ala Pro Vol Vol 185 190 195 Ala Pro Ala Pro Ala Pro Ala Pro Giu Vol Ala Thr Lys His Phe Thr Leu Lys zoo 205 210 215 Ser Asp Vol Leu Phe Asn Phe Asn Lys Ala Thr Leu Lys Pro Glu Gly Gin Gin 220 Z25 230 Ala Leu Asp Gin Leu Tyr Thr Gin Leu Ser Asn Met Asp Pro Lys Asp Gly Ser 235 240 245 250 Ala Vol Vol Leu Gly Tyr Thr Asp Arg Ile Gly 5cr Glu Ala Tyr Asn Gin Gin 255 260 265 270 Leu Ser Glu Lys Arg Ala Gin 5cr Vol. Vol Asp Tyr Leu Vol Ala Lys Gly Ile 275 280 285 Pro Ala Gly Lys Ile Ser Ala Arg Gly Met Gly Giu Ser Asn Pro Vol Thr Gly 290 295 30O 305 Asn Thr Cys Asp Asn Vol Lys Ala Arg Ala Ala Leu Ile Asp Cys. Leu Ala Pro 310 315 320 Asp Arg Arg Vol Giu Ile Glu Vol Lys Gly Tyr Lys Giu Vol Vol Thr Gin Pro 325 330 335 340 Gin Gly 41 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: Length: 567 base pairs Type: nucleic acid Strandedness.: single Topology: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: LOCATION: l.*.567 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG AAA Met Lys 1 TAT GCA Tyr Ala TAC GGT Tyr Gly CTT GGT Leu Gly TTC GAA Phe Glu GTT GAC Val Asp CTG GGT Leu Gly GCA ATT Ala Ile GGT GGT Gly Gly 29 AAC GGT Asn. Gly GCT GGT Ala Gly ATG GGT Met Gly AAC GGT Asn Gly TAC CCG Tyr Pro 100 CTG AAT GCG GCT Leu Asn Ala Ala 19 GGT TGG TCC CAG Gly Trp Ser GlIb 25 AAC AAC AAC GGT Asn Asn Asn Gly 40 GGT GGT TAC CAG Gly Gly Tyr Gln TGG CTG GGC CGT Ti-p Leu Gly Arg AAA GCT CAG GGC Lys Ala Gin Gly GAC GAT CTG GAC Asp Asp Leu Asp 105 GAT AAC ACC TGG Asp Asn Thi- Ti-p GAC ACC GGT iTC Asp Thr Gly Phe CGT AAC GAT CAG Arg Asn Asp Gin CCG TAC CTC GGT Pro Tyr Leu Gly TAT AAA GGC AGC Tyr Lys Gly Sei- CTG ACC GCT AAA Leu Thi- Ala Lys ACC CGT CTG GGC Thr Arg Leu Gly 110

GGC

Gly

GTT

Val

GGC

Gly 145

CAG

.Gl n

CAT

Asp ATC GTT TGG CGC GCT GAC TCC Met Vol Trp Ae-g Ala Asp Ser 115 120 TCC CGT AGC GAA CAC GAC ACT Ser Arg .Ser Glu His Asp The- 130 135 GTA GAG TGG GCT GTt ACT CGT Val Clu TpAlo Val The- Arg 150 TGG GTT AAC AAC ATC CCC GAC Trp, Vol Asn Asn Ile Gly Asp 165 AAC CCC ATG CTC AGC CTG GCC Asn Gly Met Leu See- Leu Gly 180 -42 AAA GGC AAC TAC GCC TCT ACC Lys Gly Asn Tyr Ala See- Thr 125 GGC CIT TCC CCA GTA TTT GCT Gly Vol See- Pro Vol Phe Ala 140 GAC ATC CCT ACC CGT CTC GAA Asp Ile Ala The- Arg Leu Glu 155 CCG GCC ACT GTG CCI ACC CCI Ala Gly Thr Vol Gly Thr Arg 170 175 CIT TCC TAC CGC TAA Vol See- Tyr Ae-g 185 INFORM~ATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: Length: 188 amino acids Type: amino acid Strandedness: single Topology: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ala Ile Phe Vol Leu Asn Ala Ala Pro Lys Asp 10 Gly Gly Lys Leu Gly Te-p See- Gln Tyr His Asp The- Cly 25 30 Phe Gln Asn Asn Asn Gly Pro The- Ae-g Asn Asp Gln Leu 45 Cly Gly Tyr Gin Vol Asn Pro Tyr Leu Gly Phe Glu Met 60 65 NO: 6: Asn The- Te-p Tyr Ala Phe Tyr Gly Asn Gly Gly Ala Gly Ala Phe so Gly Tyr- Asp Te-p Leu 43 Gly Arg Met Ala Tyr Lys Gly 5er Val Asp Asn Gly Ala Phe Lys Ala GIn Gly so 85 Vol Gin Leu Thr Ala Lys Leu Gly Tyr Pro Ile Thr Asp Asp Leu Asp le Tyr 180 105 Thr Arg Leu Gly Gly Met Vol Trp Avg Ala Asp Ser Lys Gly Asn Tyr Ala Ser 110 115 .120 125 Thr Gly Vol Ser Avg 5er Glu His Asp Thr Gly Vol Ser.Pro Val Phe Ala Gly 130 135 149 Gly Vol Glu Trp Ala Vol Thr Arg Asp Ile Ala Thr Arg Leu Giu Tyr Gin Trp 145 15@ 155 160 Vol Asn Asn Ile Gly Asp Ala Gly Thr Vol Gly Thr Avg Pro Asp Asn Gly Met 165 170 175 180 Leu Ser Leu Gly Vol 5er Tyr Avg 185 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: Length: 774 base pairs Type: nucleic acid Strandedness: single Topology: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: l.*.774 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATG AAA GCA A17 TTC GTA CTG AAT GCG CAA CAC GAT GAA GCC GTA GAC 48 Met Lys Ala Ile Phe Vol Leu Asn Ala Gin His Asp Giu Ala Vol Asp 1 5 10 GCG AAT TTC GAC CAA TIC AAC AAA TAT GGA GTA ACT GAC TAT TAC AAG 96 Ala Asn Phe Asp Gin Phe Asn Lys Tyr Gly Vol Ser Asp Tyr Tyr Lys 25 44 GTT GAA GCC AAT CIA ATC MAC AAT GCC AAA ACT GTA MAA GAC C77 CAA Asn Leu Ile Asn Asn Ala Lys Thr Val Giu Gly Val Lys Asp Leu Gin GCA CAA Ala Gin CAT GC Asp Gly GTT AAA .Val Lys CUT CAC Leu Asp GCC AAA Ala Lys TCA GC Ser Ala 130 TTlC TTC Phe Leu 145 TTA GCT Leu Ala GTA ACT Vol Ser GCT CTA Cly Val GAC ACT Asp Thr I 10 CIT CUT GAA VOl .Val Glu TTA TCT CAT Leu Ser Asp TCA AUT CAA Ser Ile Clu AAA TAT GGA Lys Tyr Gl~y ACT GUT GAA Thr Val Glu 11-5 AAG AAA CCG Lys Lys Ala AAA TCA CAA Lys 5cr Gin GAA GCT AAA Glu Ala Lys 165 GAC TAT TAC Asp Tyr Tyr 180 AAA CCA CTC Lys Ala Leu 195 TAC AAA TTA Tyr Lys Leu TCA GC AAG 5cr Ala Lys 55 TIC TTG AAA Pfi' Leu Lys 70 TTA GCT-GAA Leu Ala Glu GTA ACT GAC Vol Ser Asp CCI CIA A Cly Val Lys 120 CGT AUT TCA Arg Ile 5cr 135 ACA CCT GCT Thr Pro Ala 156 CIC iTA GCC Val Leu Ala AAG AAC CTA Lys Asn Leu ATA GAT GAA Ile Asp Clu 200 ATC CUT AAT Ile Leu Asn UiS AAA GC CCT Lys Ala Arg TCA CAA ACA Ser Cmn Thr 75 GCT AAA CTC Ala Lys Val 90 TAT CAC AAG Tyr His Lys 105 GAC CUT CAA Asp Leu Gin GAA CCA ACA Clu Ala Thr GAA CAT ACT Ciu Asp Thr 155 AAC AGA GMA Asn Arg Glu 170 ATC AAC AAT Ile Asn Asn 185 ATT TTA GC le Leu Ala GCT AAA ACA Cly Lys Thr AUT TCA GAA CCA ACA le Ser Glu Ala Thr CCI GCC CAA CAT ACT Pro Ala Clu Asp Thr TrA GCT AAC AGA GAA Leu Ala Asn Arg Clu AAC CIA ATC AAC MIT Asn Leu le Asn Asn 110 CCA CAA CUT CUT AA Ala Gin Vol Vol Clu 12S GAT CCC TTA TCT CAT Asp Gly Leu Ser Asp 140 CUT AAA TCA AUT CAA Val Lys Ser le Ciu 160 CU CAC AAA TAT GGA Leu Asp Lys Tyr Cly 175 CCC AAA ACT CUT GAA Ala Lys Thr Val Clu 190 GCA UTA CCT AAC ACT Ala Leu Pro Lys Thr 205 UCG AAA CCC GAA ACA Leu Lys C'Iy Clu Thr 45 ACT ACT GAA GCT GTT CAT GCT GCT ACT GCA AGA TCT TTC AAT TTC CCT 720 Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Arg Sei" Phe Asn Phe Pro 225 230 235 240 ATC CTC GAG AAT TCC CGG GGA TCC GTC GAC CTG CAG CCA AGC TTA AGT 768 Ile Leu Glu Asn Ser Arg Gly Ser Vol Asp Leu Gln Pro Ser Leu Ser 245 250 255 AAG TAA Lys INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: Length 257 amino acids Type amino acid Strandedness single Topology :linear (ii) MOLECULE TYPE :protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Lys. Ala Ile Phe Val Leu Asn Ala Gin His Asp Giu Ala Val Asp Ala Asn 1 5 10 Phe Asp Gin Phe Asn Lys Tyr*Gly Vol Sen Asp Tyr Tyr Lys Asn Leu le Asn Z5 30 .Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gin Ala Gin Vol Val Giu Ser 45 Ala Lys Lys Ala Arg Ile Ser Giu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys 60 65 Ser Gin Thr Pro Ala Glu Asp Thr Vol Lys Se Ile Giu Leu Ala Giu Ala Lys 80 85 Val Leu Ala Asn Arg Glu Leu Asp iys Tyr Gly Vol Sen Asp Tyr His Lys Asn 100 105 Leu Ilie Asn Asn Ala Lys Thr Vol Glu Giy Val Lys Asp Leu Gin Ala Gln Vol 110 115 120 46 Val Phe 145 Gi u Tyr

ASP

Gl y Arg 235 Pro Glu Leu Ala Lys Gl u 200 Lys Ser Se r Ala Arg Pro Ala Asn Arg 170 Asn Ala Leu Pro 205 Glu Thr Ile Leu Glu Thr 155 Asp Val Asp Glu 5cr 245 Asp Gly 140 Set' Ile Gly Vol Vol Lys 195 Lys Leu Asp Ala 230 Ser Vol Leu Ser Glu Leu 160 Set Asp Ala Leu Ile Leu 215 Ala Thr Asp Leu 250

Claims (16)

1. 8. Immunogenic complex according to one of Claims 1 to 7, characterized in that it comprises at least one unit: aD galactose p(l-2)-aD mannose p(1-4)-aL Rhamnose p(l-3) I ctyvelose p(1-3).
2. 9. Immunogenic complex according characterized in that it comprises oligosaccharide of formula C a-Tyvp(1-3) to Claim at least 8, one *0* o o 000 aD-Galp(1-2) D-Manp(l-4) aL-Rhap(1-3) in which Gal represents galactose Man represents mannose Rha represents rhamnose Tyv represents tyvelose and n may vary between 1 and 24.
3. 10. Immunogenic complex according to one of Claims 1 to 9, characterized in that the carrier protein has a) the seq ID No. 2, ID No. 4 or ID No. 6, or b) a sequence having at least 80% homology with one of the sequences mentioned in a).
4. 11. Immunogenic complex according to one of Claims 1 to 10, characterized in that it consists of at least one oligosaccharide of formula c -Tyvp (1-3) aD-Galp(1-2) aD-Manp(1-4) aL-Rhap(l-3) n in which Gal represents galactose Man represents mannose 49 Man represents mannose Rha represents rhamnose Tyv represents tyvelose and n may vary between 1 and coupled with a protein having one of the sequences ID No. 2, ID No. 4, ID No. 6 or ID No. 8, or having at least 80% similarity with one of the sequences ID No. 2, ID No. 4 or ID No. 6 or ID No. 8.
5. 13. Immunogenic complex according to one of Claims 1 to 3, characterized in that it comprises at least one Salmonella capsular antigen.
6. 14. Use of an immunogenic complex according to one of Claims 1 to 13, for the preparation of a vaccine. Use of a complex according to Claim 12, for the preparation of a vaccine intended for protecting an animal against the infections caused by Salmonella bacteria belonging to the antigenic serogroup 0:9.
7. 16. Vaccine characterized in that it comprises an immunogenic complex according to one of Claims 1 to 12 and in that it comprises, in addition, a Salmonella capsular antigen.
8. 17. Method of preparing an immunogenic complex according to one of Claims 1 to 12, characterized in that: a) Salmonella oligosaccharides are isolated from membrane lipopolysaccharides, b) optionally, the oligosaccharides are purified so as to keep oligosaccharides of the same molecular weight, c) the oligosaccharides are chemically activated, d) the activated oligosaccharides are coupled to a carrier protein in order to form the immunogenic complex.
9. 18. Method according to Claim 17, characterized in that the carrier protein is activated before step d) by a chemical method in order to facilitate the coupling.
10. 19. Method according to either of Claims 17 and 18, characterized in that an additional step is then D/003709783 50 carried out which consists of coupling the complex obtained at the end of step d) with another Salmonella antigenic determinant. 19. A method for enhancing the immunogenicity of a gram-negative bacterium oligosaccharide including the step of forming a complex between said oligosaccharide and a protein having one of the sequences ID No. 2, 4 or 6. Immunogenic complexes of claims 1 to 12 and 14 as described herein with reference to the examples.
11. 21. Use of immunogenic complexes of claims 1 to 12 and 14 described herein with reference to the examples.
12. 22. A method of preparing immunogenic complexes of claims 1 to 12 and 14 as described herein with reference to the examples.
13. 23. Use of a complex according to claim 14 in which the animal is a mammal (including a human).
14. 24. A method of protecting an animal against infections caused by bacteria having at least one oligo- or polysaccharide epitope comprising the step of administering an immunogenic complex according to any one of claims 1 to 12 with a pharmaceutically acceptable carrier to the animal. A method according to claim 24 in which the animal is a mammal (including a human). a. S.
15. 26. A method for protecting an animal by vaccination a. 25 against infections caused by Salmonella bacteria belonging to the antigenic serogroup 0:9 by administration of a complex oooo according to claim 11 with a pharmaceutically acceptable ocarrier to the animal.
16. 27. A method according to claim 26 in which the animal 30 is a mammal (including a human). Pierre Fabre Medicament SBy its registered patent attorneys SFreehills Carter Smith Beadle 15 March 2001