Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0275—Salmonella
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6068—Other bacterial proteins, e.g. OMP
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to new immunogenic complexes comprising a saccharide derivative, useful in particular as medicaments and in particular as vaccines.
• Proteins and polysaccharides are the two main types of surface antigens found in bacteria and fungi and are, by their antigenic nature, excellent tools that can be used in the design of vaccines.
• the development of defined vaccines devoid of side effects requires the use of low molecular weight vaccinating antigens, mainly peptides or oligosaccharides.
• antigens but also others of higher molecular mass such as polysaccharides, cannot alone induce an immune response which is intense and lasting.
• bacterial po saccharides in the preparation of vaccines appeared at the start of the 20th century. These compounds play an important role in the structure and pathogenicity of certain Gram positive and negative bacteria.
• Two types of poh bacterial saccharides are excellent candidates as a vaccine agent. These are polysaccharides from the bacterial capsule and polysaccharides from lipopoh saccharides (LPS) from the outer membrane of Gram negative bacteria.
• polymers essentially composed of a carbohydrate part, and part of the molecule of which is exposed on the surface of the bacteria. They consist of a linear sequence of repeating units, characteristic of a given bacterial species, the number of which can vary from one to several hundred, thus explaining their molecular weight which is sometimes very high.
• Each repeating unit itself consists of several monosaccharides linked together by glycosidic bonds, generally from 1 to 7 monosaccharide residues. The latter can be more or less substituted by mineral groups such as phosphates or by organic groups, such as acids, amino, alcohols, fatty acids or amino acids.
• Vaccines comprising a polysaccharide extract have been shown to be relatively effective in adults at low doses, 25 to 50 ⁇ g.
• vaccines based on bacterial polysaccharides, there are to date vaccines against infections
• Neisseria meningitidis tetravalent vaccine against strains of groups A, C, YV135 and Y
• Streptococcus pneumoniae multivalent pneumococcal vaccine combining 23 serotypes of capsular polysaccharides
• Salmonella typhi vaccine composed of the capsular polysaccharide of S. typhi.
• Salmonella typhi vaccine composed of the capsular polysaccharide of S. typhi.
• 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, pathogenic for humans and for many vertebrate animals. The genus Salmonella is conventionally made up of more than 2000 different serotypes. The main pathologies induced by these bacteria are (for general reviews see: Le Minor, L, 1987, Salmonella in Medical Bacteriology, L Le Minor and M. Véron Eds., Médecine-Sciences Flammarion Paris, p. 41 1- 427, Pegues, DA and Miller, SI, 1994, Salmonellosis including typhoid fever, Curr. Opin. Infect Dis. 7, 616-623): - in animals, toxi
• Salmonella abortus-ovis in sheep Salmonella galinarum in poultry
• typhoid fevers (Salmonella typhi) and paratyphoid fevers (Salmonella paratyphi A, B and D),
• the "inactivated bacterial vaccine” type injectable forms are the oldest vaccine forms. These are vaccines that can contain 500 to 1000 million bacteria per dose, in liquid form. The bacteria are inactivated by heat treatments and / or by chemical compounds such as acetone, formalin or phenol. Depending on the bacterial serotypes they contain, there are
• this second class includes "Vac TAB” vaccines from PASTEUR in France or "Typhidrall” from BIOCINE SCLAVO in Italy.
• the TAB (PASTEUR) vaccine is a complete inactivated trivalent liquid bacterial vaccine, combining Salmonella typhi, Salmonella paratyphi A and B. Its relative efficacy is in fact limited to the valence T. This vaccine is reactogenic: it causes in a third cases of early local and general reactions. 11 is injected subcutaneously, 2 to 3 injections, 2 to 4 weeks apart, followed by a booster. In France the current regulations which still impose it on certain professional categories, including the military and health professions, are in fact no longer justified. In the military, we now use a monovalent T vaccine, not marketed.
• Ty21a oral typhoid vaccine (“Vivotif", BERNA) contains a living defective strain of Salmonella typhi, devoid of galactose-4-epimerase. Its administration is oral, in the form of gastroresistant capsules containing 1 to 8.10 9 live bacteria in lyophilized form.
• the vaccination schedule includes 3 successive doses on days 1, 3 and 5, with simultaneous intake of baking soda to neutralize stomach acid. Evaluated in endemic areas in Egypt and Chile, the protective value would be between 60 and 90%. There would be no side effects. Vaccine protection becomes effective approximately 10 days after the last dose. The protection is valid for a period ranging from 1 to 7 years, and it is therefore recommended that travelers to endemic regions repeat the vaccination annually. Older oral forms are still available. These are inactivated vaccines, the efficacy of which, however, has not been clearly established (“Taboral" from the company BERNA, "Enterovaccino” in Italy).
• the MERIEUX Institute's "Typhim Vi” vaccine is prepared from purified Vi capsular polysaccharide of Salmonella typhi. It is a solution for injection containing 25 ⁇ g of polysaccharide.
• a single injection, subcutaneous or intramuscular, provides protection only against the infectious risk associated with Salmonella typhi in adults and children over 5 years of age.
• This vaccine does not provide protection against Salmonella paratyphi A and B, these serotypes not being encapsulated. Immunity appears approximately 15 days to 3 weeks after the injection. The term of protection is at least 3 years. In high endemic areas, the protection rate observed is around 60%.
• immunogenic complexes characterized in that they consist of at least one oligo- or polysaccharide epitope naturally present in pathogens such as bacteria, coupled to a carrier protein chosen from the binding protein to human serum albumin from Streptococcus, the outer membrane proteins of gram negative bacteria, or their fragments. Indeed, the coupling by a covalent bond of the oligosaccharide or of the polysaccharide transforms the latter into T-dependent immunogen.
• the oligo- or polysaccharide epitope is capable of being obtained from bacteria, gram negative or gram positive, and in particular from membrane lipopolysaccharides or capsule oligosaccharides, bacteria of the genus Salmonella, Escherich ia, Neisseri a, Shigel la, I laemophilus or Klebsiella.
• the pathogenic agent can be Salmonella typhi, Haemophilus influenzae, Neisseria miningitidis or Streptococcus pneumoniae.
• the oligo- or polysaccharide epitope can 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 the LPS from the membrane and then removing lipid A by gentle hydrolysis.
• the capsular polysaccharides are more easily isolated from the bacterial suspension: heating at 100 ° C. for ten minutes (solubilization) followed by centrifugation makes it possible to isolate the capsular polysaccharide Vi from Salmonella typhi.
• the two types of polysaccharides can then be purified by chromatography and / or by membrane filtration.
• the use of "whole" polysaccharides in a coupling process can see certain technical problems appear, mainly due to the too large size of these compounds: gel formation, precipitation.
• cleavage methods of the polysaccharide can be used: ultrasonic fragmentation, depolymerization by oxidation-reduction, hydrolysis carried out in acidic or basic medium, enzymatic hydrolysis.
• An oligossacharide is a compound, which can be derived from a polysaccharide, and which, compared to the starting polysaccharide, has a reduced number of repeat units.
• conjugate vaccine on the market is a vaccine to prevent invasive Haemophilus influenzae type b infections (meningitis) in humans.
• conjugate 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 conjugate vaccine are of two types:
• TT Tetanus
• DT diphtheria
• Tetanus and diphtheria toxoids are currently the best characterized carrier proteins, both from a structural and biological point of view (vaccine properties, properties of carrier proteins), and are therefore considered to be the reference carrier proteins.
• TT toxin is a 150kDa protein.
• the DT toxin has a lower molecular weight and is secreted as a single polypeptide chain of 535 amino acids. After purification, these proteins are inactivated by heat and formalin. They can be combined with each other (DT vaccine), and with many other vaccines (whooping cough, poliomyelitis ). Heat resistant, they keep at + 4 ° C for a few years, but should not be frozen.
• 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 actually contains several proteins associated with lipids and lipopolysaccharides.
• the carrier protein comprises at least part of an OmpA type protein of gram negative bacteria, in particular of an outer membrane protein of Klebsiella pneumoniae.
• Proteins particularly suitable for carrying out the present invention are proteins derived from the major membrane protein of Klebsiella pneumoniae 1- 145, hereinafter designated p40; they present in particular one of the sequences ID No. 2, ID No. 4 or ID No. 6.
• Other proteins of interest derived from the outer membrane protein of K. Pneumoniae include fragments
• Invariable extramembrane loops are defined as homologous P40 sequences with the loop sequences conserved between different species of enterobacteria.
• the sequences of extramembrane loops not conserved during evolution are called variable loops.
• the localization of the extramembrane loops is carried out according to the model of VOGEL and JAHNIG (1986, J. Mol. Biol., 190: 191-199) concerning the OmpA of E. coli.
• the fragments between amino acids 127 to 179 of sequence ID No. 1 will be used.
• Suitable sequences are respectively the sequences between amino acids 108 to 179 of sequence ID no . 1, amino acids 1 to 179 of sequence ID no. 1, as well as sequences having at least 90% of homology with the preceding sequences.
• the carrier protein comprises all or part of the human serum albumin binding domain of the streptococcus protein G (hereinafter called BB).
• BB streptococcus protein G
• This protein has a molecular mass of 29 kDa and can 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 80%, and preferably at least 90% of similarity with said sequence ID no. 8.
• All of these carrier proteins can be extracted from the original bacteria or else obtained by the recombinant DNA pathway.
• Immunogenic complexes 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 bacteria of the genus Salmonella will belong to a 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 .
• the immunogenic complex contains an oligosaccharide capable of being obtained from the lipopolysaccharide of Salmonella enteritidis with antigen specificity 0: 9.
• Salmonella serotypes are identified by their antigenic formula; they are classified into different groups, according to their antigenic specificity O.
• Salmonella typhi belongs to group D, with specificity 0: 9, as well as S. enteritidis, S. panama, and S. dublin.
• - serogroup B of specificity 0: 4, having as representative t S. paratyphi B and S. typhimurium
• - serogroup C of specificity 0: 6, 7, 8, having as representative S. infantis and S. bovis morficans ,
• oligosaccharides belonging to the major antigenic specificities listed above will be particularly suitable for the implementation of the invention.
• a vaccine prepared from oligosaccharide isolated from S. enteritidis lipopolysaccharide, carrying the antigen specificity 0: 9, will protect against septicemia with Salmonella typhi and against typhoid fever, but it can also be used in prevention in humans and animals of toxi- infections and zoonoses due to Salmonella from the same serogroup.
• Oligosaccharides according to one of the preferred aspects of the invention have at least one unit: ⁇ D galactose p (l-2) - ⁇ D mannose p (l-4) - ⁇ L Rhamnose p (l-3)
• the subject of the invention is an immunogenic complex comprising at least one oligosaccharide of formula
• Man represents mannose Rha represents rhamnose Tyv represents tyvélose and n can vary between 1 and 24.
• n can vary between 1 and 5, and the oligosaccharide is coupled with a protein having one of the sequences ID n "2, ID n" 4, ID n "6, or ID n ° 8, or having at least at least 80% similarity with one of the sequences ID No. 2, ID No. 4 or ID No. 6 or ID No. 8.
• the invention also relates to the use of immunogenic complexes as defined above, for the preparation of a vaccine; according to a particularly advantageous aspect, the complexes are useful for the preparation of a vaccine intended to protect an animal against infections caused by the Salmonella bacteria belonging to the 0: 9 antigenic serogroup.
• n will have different values.
• Immunogenic complexes according to the invention are also those comprising a capsular Salmonella antigen.
• the latter can be coupled alone to a carrier protein as defined above, or else associated with a complex comprising another oligo- or polysaccharide epitope.
• a 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 adjuvants of imm unity, and pharmaceutically acceptable excipiens ts necessary for their formulation such as diluent, stabilizer, preservatives, etc., known to those skilled in the art.
• the invention relates to a vaccine containing a membrane oligosaccharide coupled to a carrier protein, and further comprising another antigenic determinant.
• the vaccine comprises a capsular antigen of Salmonella, such as the capsular antigen Vi (homopolymer of partially acetylgalacturonic acid N); this increases the effectiveness of the vaccine against the encapsulated bacteria.
• the process for the preparation of the unogenic immunogenic complex can comprise the following stages: a) oligosaccharides from Salmonell are isolated from membrane lipopolysaccharides, b) optionally, the oligosaccharides are purified so as to preserve oligosaccharides of the same molecular weight , c) the oligosaccharides are chemically activated, d) the activated oligosaccharides are coupled to a carrier protein to form the immunogenic complex.
• the carrier protein is activated before step d) by a chemical method to facilitate coupling.
• oligosaccharides of different sizes can be envisaged. These oligosaccharides being released by enzymatic cleavage (endorhamnosidasic activity of a phagc), it will preferably be multiples of 4: tetrasaccharides, octasaccharides, dodecasaccharides, hexadecasaccharides, icosasaccharides ... Similarly the coupling of a mixture of these oligosaccharides (without prior purification) is included in the invention.
• step d) it is then possible to carry out an additional step consisting in coupling the complex obtained at the end of step d) with another antigenic determinant of Salmonella.
• the invention also relates to the use of a protein comprising one of the sequences ID No. 2, 4, 6 or 8 to improve the immunogenicity of an oligosaccharide.
• analogous proteins in which at least one amino acid has been replaced by a homologous amino acid in 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, 5 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
• sequences ID No. 5 and 6 correspond to the entire transmembrane part, ( ⁇ P40F8), and are devoid of the very immunogenic periplasmic part (Puohiniemi, R., Karvonen, M., Vuopio-Varkila, J., Muotiala, A., Helander, IM and Sarvas, M., 1990, Infect. Immun. M, 1691-1696)
• sequence ID No. 8 corresponds to the human serum albumin binding domain of the Streptococcus protein G.
• Figure 1 Demonstration of the immunogenic power of the P40-icosasaccharide conjugate in mice.
• Figure 2 Demonstration of the immunogenic power of the P40-icosasaccharide conjugate in rabbits.
• EXAMPLE 1 Isolation and purification of the natural P40 protein.
• the P40 protein a major protein of the outer membrane of Klebsiella pneumoniae is isolated by extraction in the presence of a detergent from the biomass Klebsiella pneumoniae, then purified from the extract thus obtained by anion exchange chromatography then cations.
• the pH of the Klebsiella pneumoniae biomass (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 adding 0.5 volume of a solution containing
• the supernatant is an extract of membrane proteins from Klebsiella pneumoniae.
• the supernatant proteins are precipitated by adding 3 volumes of 95 ° ethanol at -20 ° C (final ethanol concentration ⁇ 80%). After rapid stirring, the whole is left to stand for 1 hour at 4 ° C minimum. The precipitated proteins are collected by centrifugation for 10 min at 10000 g at 4 ° C.
• the pellets are resuspended in a 1% solution of Zwittergent 3- 14 at a rate of 5 ml / g of wet pellet. After stirring for 1 hour (propeller stirrer) and grinding using an ultra-turrax (13500 rpm, 30 sec), the pH is adjusted to 6.5 using I N sodium hydroxide. Centrifugation of the mixture makes it possible to obtain the fraction MP (elimination of the insoluble matter).
• the proteins of the MP are dialyzed overnight at 4 ° C. against a Tris / HCl buffer 20 mM pH 8.0, 0.1% Zwittergent 3-14.
• the dialysate is deposited on a column containing a support of the strong anion exchanger type (Biorad Macro Prep High Q gel) balanced in the buffer described above at a linear flow rate of 15 cm / h. Proteins are detected at 280nm.
• the P40 protein is eluted, with a linear flow rate of 60 cm / h, for a concentration of 0.2M NaCl in the Tris / HCl buffer 20mM pH 8.0; 0.1% Zwittergent 3-14.
• the fractions containing the P40 protein are combined and concentrated by ultrafiltration using an Amicon shaking cell system used with a Diaflo membrane of the YM 10 type (cutoff threshold 10kDa) for volumes of the order of 100 ml, or using a Minitan Millipore tangential flow filtration system used with membrane plates with a cut-off threshold of OkDa for higher volumes.
• the fraction thus concentrated is dialyzed overnight at 4 ° C against a 20mM citrate buffer pH 3.0, 0.1% Zwittergent 3- 14.
• the dialysate is deposited on a column containing a support of strong cation exchanger type (Biorad Macro Prep High S gel) balanced in the citrate buffer 20mM pH3.0, 0.1% Zwittergent 3-14.
• the P40 protein is eluted (speed 61 cm / h) for a 0.7M NaCl concentration.
• the fractions containing P40 are combined and concentrated as described above.
• the fractions obtained after each chromatographic step are analyzed by SDS PAGE in order to gather those containing the P40 protein.
• the amounts of protein are determined by assay according to the Lowry method.
• the purity and homogeneity of the P40 protein are estimated by SDS PAGE with revelations in coomassie blue and silver nitrate.
• the P40 is concentrated in order to reach a protein concentration of the order of 5 to 10 mg / ml.
• the electrophoretic profiles reveal a degree of purity greater than 90%.
• the protein is specifically recognized by an anti-P40 monoclonal antibody obtained in mice.
• the nucleotide primers were determined from the part of the published sequence of the OmpA of Klebsiella pneumoniae LD 199 (Lawrence JG et al., 1991, J. Gen. Microbiol. 131, 191 1-1921), of the sequence conscience based on the alignment of the OmpA sequences of different enterobacteria (Escberichia coli, Salmonella typhimurium, Serratia marcescens, Shigella dyscnteriae, Enterobactcr aeroginosae) as well as sequences of peptides obtained by sequencing according to the Edman method of the natural protein isolated from Klebsiella pneumoniae 1- 145 and peptides isolated after digestion with cyanogen bromide.
• the oligonucleotides were synthesized by the chemical method of phosphoramidites using the Pharmacia Gene Assembler Plus device.
• a colony of Klebsiella pneumoniae 1- 145 is lysed in 10 ⁇ l of lysis buffer (25mM Taps pH 9.3, 2mM MgC12). 1 ⁇ l of this solution is then used as a DNA source for the PCR amplification reactions. These are carried out in 100 ⁇ l of amplification buffer with 5 pmol of each primer and an enzymatic unit of Taq polymerase (Perlcin Elmer Cetus). Each cycle includes a 30 second denaturation step at 95 ° C followed by hybridization of the primer with DNA and an extension of one minute at 72 ° C.
• the fragment thus cloned is sequenced using an Applied Biosystem 373 DNA Sequencer automatic sequencer.
• the sequencing reactions are carried out using the dye terminator kit according to the supplier's recommendations.
• the entire gene for the P40 protein is then cloned into the expression vector pTrp inducible by the presence of the gene for the operon tryptophan, carrying the gene for resistance to kanamycin and having two restriction sites Bsml and Sali.
• a Bsml restriction site is introduced by PCR upstream of the P40 gene, which already has a SalI site downstream, in the vector pRIT28P40.
• the P40 gene having Bsml / SalI sites is thus cloned into the vector pTrp to constitute the plasmid pTrpLP40.
• the fusion protein LP40 is expressed and produced in
• Escherichia coli as an inclusion body. This will include the complete sequence of Klebsiella pneumoniae OmpA to which must be added at the N-terminal end an 8 amino acid peptide (L peptide) comprising part of the leader sequence of the tryptophan operon necessary for the expression of the protein in Escherichia coli.
• L peptide 8 amino acid peptide
• the expression of the protein LP40 is carried out in Escherichia coli
• RRI ⁇ M15 (R ⁇ ther, U., 1982, Nucl. Acid Res. Ifl 5765-5772).
• a preculture is carried out with stirring at 37 ° C. overnight in a medium based on soy tryptic broth (TSB medium) supplemented with yeast extract and in the presence of kanamycin 30 ⁇ g / ml.
• TLB medium soy tryptic broth
• the vector operating region is blocked in the presence of an excess of tryptophan (100 ⁇ g / ml).
• the culture After reading the optical density at 580nm, the culture is diluted in order to obtain an optical density of 1 in the preceding medium (TSB medium with yeast extract and kanamycin).
• TLB medium with yeast extract and kanamycin.
• the synthesis of the protein LP40 is induced by the addition of indolacrylic acid (analog of tryptophan) at the final concentration of 25 ⁇ g / ml.
• the culture is maintained at 37 ° C. with stirring for 5 hours. 2.1.4. Renaturation and purification of the LP40 protein.
• the cells After centrifugation (4000 rpm, 10 min, 4 ° C), the cells are resuspended in a 25 mM Tris MCI buffer pH 8.5. Sonication allows the release of inclusion bodies.
• the inclusion body pellet obtained by centrifugation (25 min at 10000 g at 4 ° C) is taken up in a 25 mM Tris-HCl buffer pH 8.5 containing 5 mM MgCl 2 , then centrifuged (15 min at 10000 g). 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 7M guanidinium hydrochloride or urea (denaturing agent) and OmM dithiothreitol (reduction of disulfide bridges). Centrifugation (1 5 min at 10000 g) eliminates the insoluble part of the inclusion bodies.
• the sample is dialyzed against a 25 mM Tris-HCl buffer pH 8.5 containing 0.1% Zwittergent 3-14 (100 volumes of buffer) overnight at 4 ° C.
• the anion exchange chromatography step is carried out on the Biorad Macro Prep High Q support as described above (Example 1).
• the fractions containing the LP40 protein are combined and then concentrated by ultrafiltration before a new dialysis against a 20mM citrate buffer pH 3 containing 0.1% Zwittergent 3-14 (100 volumes of buffer) overnight at 4 ° C.
• the cation exchange chromatography step is carried out on the Biorad Macro Prep High S support as described in Example 1.
• the fractions containing the LP40 are combined and then concentrated by ultrafiltration.
• the Klebsiella pneumoniae (P40) OmpA gene contains 1008 base pairs (Sequence ID No "1) and codes for a protein of 335 amino acids (Sequence ID No 2).
• the LP40 gene has 1035 base pairs (Sequence ID No.3) and codes for a protein of 344 amino acids (Sequence ID No.4).
• As regards the part of the OmpA gene there are some differences at the end coding for the two amino acids in the C-terminal position. These differences in fact concern only three nucleotides.
• a 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 the two chromatographic steps.
• the LP40 protein is concentrated after purification in order to reach a final concentration of between 5 and l Omg / ml.
• the electrophoretic profiles show a degree of purity of the order of 95%.
• the protein is specifically recognized by a natural anti-P40 monoclonal antibody obtained in mice.
• the state of the protein is followed by SDS-PAGE.
• the P40 protein extracted from the membrane of Klebsiella pneumoniae has a characteristic electrophoretic (migration) behavior.
• the native form indeed has a lower molecular weight than the denatured form ( ⁇ -helix structure) under the action of a denaturing agent, such as urea or guanidinium hydrochloride, or by heating to 100 ° C in the presence of SDS.
• the protein LP40 is not correctly renatured at the end of renaturation, whether this is carried out in the absence or in the presence of 0.1% (w / v) Zwittergent 3- 14.
• EXAMPLE 3 Cloning, expression and purification of the transmembrane part of the P40 protein.
• the gene sequence corresponding to the fragment sought was amplified by PCR from the DNA of a miniprep of the vector pRIT28P40, then purified and cloned in the same vector. A sequencing is carried out in order to verify that no mutation has occurred during the amplification.
• This gene is then cloned as described above (Example 2) into the vector pTrp to constitute the plasmid pTrpL ⁇ P40F8.
• the fusion protein L ⁇ P40F8 is expressed in Escherichia coli
• the gene for the protein L ⁇ P40F8 comprises 567 base pairs (sequence ID no. 5) and codes for a protein of 11-18 amino acids (sequence ID no. 6).
• EXAMPLE 4 Expression and purification of the BB protein
• the BB protein gene is cloned into the pva expression vector inducible by the presence of the tryptophan operon gene, carrying genes for resistance to ampicillin and tetracycline and having an origin of replication in Escherichia coli.
• the BB protein is expressed and produced in Escherichia coli RV 308 (strain ATCC 31608) in the form of inclusion bodies.
• the competent Escherichia coli RV 308 strains are transformed by the vector pvaBB.
• a preculture is carried out with stirring at 37 ° C. overnight in a medium based on soy tryptic broth (TSB medium) supplemented with yeast extract and in the presence of tetracycline (8 ⁇ g / ml) and ampicillin (200 ⁇ g / ml).
• TLB medium soy tryptic broth
• tetracycline 8 ⁇ g / ml
• ampicillin 200 ⁇ g / ml
• the vector operating region is blocked in the presence of an excess of tryptophan (100 ⁇ g / ml).
• the culture After reading the optical density at 580nm, 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).
• TLB medium with yeast extract and tetracycline / ampicillin.
• the synthesis of the BB protein is induced by the addition of indolacrylic acid (analog of tryptophan) at the final concentration of 25 ⁇ g / ml.
• the culture is maintained at 37 ° C. with stirring for 5 hours.
• the cells After centrifugation (4000 rpm, 10 min, 4 ° C), the cells are resuspended in 25 mM Tris-HCl buffer pH 8.5. Sonication allows the release of inclusion bodies.
• the inclusion body pellet obtained by centrifugation (25 min at 10000 g at 4 ° C) is taken up in a 25 mM Tris-HCl buffer pH 8.5 containing 5 mM MgCl 2 , then centrifuged (15 min at 10000 g). Denaturation of the protein is obtained by incubation of the inclusion bodies at 37 ° C.
• the BB protein is purified by affinity chromatography on an HSA-Sepharose support (support prepared by coupling of human serum albumin on a Pharmacia gel "CNBr- activated Sepharose 4B").
• HSA-Sepharose support support prepared by coupling of human serum albumin on a Pharmacia gel "CNBr- activated Sepharose 4B"
• the unbound proteins are eluted with a 25 mM Tris / HCl buffer pH 8.5, 0.2 M NaCl, 0.05% Tween 20 and EDTA ImM.
• the BB protein retained on the support is eluted with a 0.5 M acetic acid solution, pH 2.7.
• the fractions containing the protein of interest are pooled and then concentrated by ultrafiltration.
• the BB protein gene has 774 base pairs (Sequence ID
• the protein expressed comprises from the N-terminal end (see Sequence ID No. 7):
• the protein produced has a molecular mass of approximately 29kDa (analysis by SDS-PAGE).
• EXAMPLE 5 Isolation and purification of the oligosaccharides of Salmonella enteritidis from lipopolysaccharides.
• the bacteria Salmonella enteritidis SH 1 262 are cultivated in a 10 liter fermenter at 37 ° C., at pH 7.0, with vigorous stirring, in a Ty medium.
• the cells are killed by the addition of 1% formaldehyde and are collected by centrifugation at 4000 g for 20 min at 4 ° C. After washing in PBS and a new centrifugation, carried out as above, the pellet is resuspended at a concentration of the order of 20 mg (dry weight) / ml.
• the LPS are extracted by the phenol method (Westphal 0., L ⁇ deritz O. and Bister F., 1952, Z. Naturforsch. 7, 148-155) and the aqueous phase is collected and lyophilized.
• Partially defatted LPS are prepared by hydrolysis of the phosphate and ester bonds at the lipid part (lipid A) by a treatment carried out in the presence of 0.15M sodium hydroxide at 100 ° C. for 2 hours. After centrifugation, the pH is adjusted to 3.5 and the free fatty acids are eliminated by successive extractions with chloroform. The pH is then adjusted to 7.0 before the LPS thus delipidated are dialyzed against water and finally lyophilized.
• the oligosaccharides are prepared from partially delipidated LPS using the endorhamnosidase activity associated with bacteriophage P36.
• a dialysis rod containing phage P36 dialysis beforehand against a 5mM ammonium carbonate buffer pH 7, 1, are added the LPS obtained previously in a ratio lg of LPS / 10 14 pfu of phage. Dialysis is carried out at 37 ° C against 600 to 800 ml of the preceding buffer. After 50 hours the dialysis bath is changed and the dialysis is renewed for an additional period of approximately 40 hours. The two counter-dialysis solutions are then mixed and then concentrated using a rotary evaporator. The oligosaccharides are fractionated by molecular sieving chromatography.
• the concentrated oligosaccharides are deposited on a column (2.5 x 170 cm) of Biogel P2 or P4 (Biorad, 200-400 mesh) eluted with water (flow rate 8.5 ml / h).
• the fractions containing the oligosaccharides are detected by the phenol method (+ sulfuric acid). After analysis of the fractions by thin layer chromatography, the fractions containing the various isomers are combined and then lyophilized.
• the purity of the oligosaccharides obtained is determined by nuclear magnetic resonance and mass spectrometry.
• EXAMPLE PLE 6 Coupling of ol igosaccha wrinkles isolated from lipopolysaccharides of Salmonella enteritidis to the protein P40.
• the oligosaccharides (10 to 40 mg) are dissolved in 0.5 ml of water. This oligosaccharide solution is added dropwise with stirring to 1 ml of a para-aminophenylethylamine solution diluted in water (v / v) containing 20 mg of sodium cyanoborohydride. After adjusting the pH to 8.0 using NaOH I N, the reaction mixture is left at room temperature for 24 hours. Excess reagents are removed by gelfiltration on a column of Biogel P2. The fractions containing the derived oligosaccharides are collected, concentrated to dryness using a rotary evaporator then the oligosaccharides are taken up in 3 ml of 80% ethanol.
• 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 protein (LP40 or ⁇ P40F8) in solution in a 0.1 M bicarbonate buffer pH 8.2 containing 0.1% Zwittergent 3-14 (2.3 ml, concentration of the order of 5 mg / ml) is added drop by drop with constant stirring with oligosaccharides.
• the pH is adjusted to 8.7 using IM soda and the reaction mixture is kept at room temperature for 48 hours.
• Uncoupled oligosaccharides are eliminated by a series of several dilutions and concentrations using an Amicon shaking cell equipped with a Diaflo membrane having a cutoff threshold of 30kDa.
• the conjugates obtained are dialyzed several times against 1 liter of PBS buffer containing 0.1% Zwittergent 3-14.
• the substitution rate 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 -20 ° C.
• the degree of substitution estimated by assaying the proteins and oligosaccharides is 5.3 moles of icosasaccharides / mole of P40. This value is in agreement with that established after SDS PAGE of the conjugate.
• mice females NMRI, lS20g, 6 / lot are immunized daily
• Each mouse receives a dose of 0.5 ml of the conjugate prepared in Example 3.
• the conjugates (10 ⁇ g) are injected in the presence or in the absence of complete Freud's adjuvant and the injections are carried out intraperitoneally.
• the blood samples are taken by puncture on days 0 and 35.
• the antibody responses are evaluated on the individual sera collected by the EL1SA method.
• the anti-icosasaccharide IgGs of the sera are isolated on the BSA-hexadecasaccharide support and are revealed using an anti-mouse IgG antiserum labeled with alkaline phosphatase.
• the optical density is determined at 405nm. 6.2. Results.
• the P40 / icosasaccharide conjugate When injected in the presence of complete Freud's adjuvant, the P40 / icosasaccharide conjugate allows the induction of a response directed against the significant oligosaccharide from day 35 (3 immunizations): the antibody titer is close to 1 / 1.10 5 .
• this titer is maintained when the immunizations are carried out in the absence of adjuvant.
• the anti-oligosaccharide antibody titers are in fact between 1 / 1.10 4 and 1 / 1.10 5 .
• EXAMPLE 8 Demonstration of the immunogenic power of the P40 / icosasaccharide conjugates in rabbits.
• Rabbits (New Zealand white rabbits, 2-3 kg) are immunized on days 0.14 and 28.
• the P40 / oligossacharid conjugates (10 ⁇ g) are injected into the popliteal lymph nodes in the presence (v / v) or absence of Freud's complete adjuvant.
• On days 0, 14, 28 and 56 blood samples are taken and the antibody responses are evaluated on the individual sera by the ELISA method.
• the titration plates are covered by 2 different antigens: the P40 protein and the LPS from which the oligosaccharides coupled to P40 are derived.
• Antibodies are revealed using an anti-rabbit IgG conjugate labeled with alkaline phosphatase. The optical density is determined at 405nm.
• the icosasaccharide when presented by the protein P40, induces in the presence of adjuvant of Freud an important response against the lipopolysaccharide from which it is derived: titer greater than 1 / 1.10 6 .
• the response directed against the icosasaccharide when presented by P40 is weaker than that obtained in the presence of adjuvant, but significantly greater than that induced after injection of the conjugate BSA-octasaccharide.
• FIG. 2 presents the results of the ELISA assay carried out against the LPS from which the icosasaccharide coupled to the protein P40 is derived. After 56 days the antibody titer is greater than 1/10000.
• EXAMPLE 9 Challenge experiment in mice after passive transfer.
• mice receive an intravenous injection of 0.2 ml of a hyperimmune serum obtained in rabbits after an immunization cycle carried out under the conditions previously described in Example 5 (injections in the absence of adjuvant, collection of the serum on D56).
• Salmonella enteritidis SH 2204 is carried out 2 to 3 hours after injection of the hyperimmune serum.
• the bacteria are injected into the animal intraperitoneally.
• the mice are observed up to 60 days after the injection.
• mice 60 days after challenges made by injecting doses of 1.3 and 13 times the LD50, all the mice are alive (Table 1), the dose 52 x LD50 being excessive (death of animals).
• Table 1 Challenge experiments in mice after passive transfer of a hyperimmune rabbit serum or after immunization with the P40-icosasaccharide conjugate. Determination of the percentage of live animals 60 days after intraperitoneal injection of a dose of Salmonella enteritidis bacteria.
• EXAMPLE 10 Challenge experiment in mice after immunization with the P40 / icosasaccharidc conjugate.
• mice (NMRI females, 20 g, 6 / lot) are immunized with the P40 / icosasaccharide conjugate in the absence of adjuvant as described in Example 4.
• Vaccination with the P40-icosasaccharide conjugate makes it possible to directly protect the mice against a challenge by Salmonella enteritidis. Immunizations with this conjugate indeed make it possible to increase the LD50 by a factor of at least 10 (Table 1), the LD50 can then be greater than 3.4 ⁇ 10 6 / ml.
• 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 15 CAG TAT CAC GAC ACC GGT TTC TAC GGT AAC GGT TTC CAG AAC AAC 96
• GCT TAC AAC CAG CAG CTG TCT GAG AAA CGT GCT CAG TCC GTT GTT GAC 816
• GAT GCT GCA CCG GTT GTT GCT CCG GCT CCG GCT CCG GAA GTG 624 Asp Ala Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala Pro Glu Val 195 200 205
• GAC ACT TAC AAA ⁇ A ATC CTT AAT GGT AAA ACA ⁇ G AAA GGC GAA ACA 672 Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr 210 215 220 ACT ACT GAA GCT G ⁇ GAT GCT GCT ACT GCA AGA TCT ⁇ C AAT ⁇ C CCT 720 Thr Thr Glu Al ⁇ Val Asp Ala Ala Thr Ala Arg Ser Phe Asn Phe Pro 225 230 235 240

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