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
  • A61K39/02—Bacterial antigens
  • A61K39/085—Staphylococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

• the invention relates to type 5 and type capsular polysaccharides
• S. aureus strains comprise a peptidoglycan wall and optionally polysaccharides linked to the outer surface of the latter which form a more or less thick structure that is called capsule or glycocalix.
• capsular polysaccharides are formed by the assembly of repeating units whose constituents and bonds are defined and are characteristic of the bacterial species. These repetitive units contain the epitopes and structures determining the antigenicity.
• a major obstacle to the development of a capsular antigen-based vaccine against S. aureus is the low productivity, variable depending on culture conditions, of wild strains expressing T5 and T8.
• the inventors have surprisingly demonstrated that wild S. aureus strains that have been genetically engineered to produce capsular polysaccharide (PS) in large amounts can be advantageously used to produce polysaccharides of interest.
• PS capsular polysaccharide
• the inventors have indeed shown that strains of S. aureus T5 and T8, modified by genetic recombination to obtain strains overproducing capsular PS, are capable of producing in large amounts in the culture supernatant a capsular polysaccharide which, although different of wild-type PS, induces a humoral response comprising antibodies against wild-type capsular PS.
• the subject of the present invention is therefore an immunogenic composition
• a T8 and / or T5 capsular polysaccharide of a T8 and / or T5 supramatronic S. aureus strain respectively.
• said composition comprises a T5 capsular polysaccharide of a strain of S. aureus CYL1892.
• said composition comprises a capsular polysaccharide T8 of a strain of S. aureus CYL770.
• said composition comprises a T5 capsular polysaccharide of a strain of S. aureus CYL1892 and a capsular polysaccharide of T8 of a S. aureus CYL770 strain.
• said immunogenic composition comprises a conjugate comprising a capsular polysaccharide T8 and / or T5 of a strain overproducing S. aureus bound to a carrier protein.
• said conjugate comprises a capsular polysaccharide T8 of strain CYL770.
• said conjugate comprises a T5 capsular polysaccharide of strain CYL1892.
• said immunogenic composition comprises a conjugate comprising a T8 capsular polysaccharide of strain CYL770 and a conjugate comprising a T5 capsular polysaccharide of strain CYL1892.
• said conjugates comprise a fractionated capsular polysaccharide.
• the carrier molecule of the conjugate according to the present invention is exotoxin A of Pseudomonas aeruginosa.
• the present invention relates to a conjugate comprising a T8 or T5 capsular polysaccharide of a strain overproducer of S. aureus linked to a carrier protein, advantageously the exotoxin A of Pseudomonas aeruginosa.
• a conjugate comprising a T8 or T5 capsular polysaccharide of a strain overproducer of S. aureus linked to a carrier protein, advantageously the exotoxin A of Pseudomonas aeruginosa.
• said PS is split.
• said conjugate comprises a capsular polysaccharide of the S. aureus strain CYL1892.
• said conjugate comprises a capsular polysaccharide of the S. aureus CYL770 strain.
• Said PS are preferably fractionated before being conjugated.
• the present invention relates to a type 5 capsular polysaccharide of S. aureus strain CYL1892. This polysaccharide is advantageously in isolated or purified form.
• the present invention relates to the use of a conjugate as defined above for the manufacture of an immunogenic composition for immunization against S. aureus infections.
• the present invention relates to a method for producing T5 capsular polysaccharide comprising the steps of: a) culturing an overproducing S. aureus strain b) inactivating the culture thus produced, and c) recovering the capsular polysaccharide T5 from the supernatant.
• the overproducing strain is the CYL1892 strain.
• S. aureus strain is understood to mean any S.aureus strain expressing a capsule or microcapsule comprising a T5 and / or T8 polysaccharide.
• S. aureus strain s
• the strains advantageously used in the context of the present invention are the Reynolds and Becker strains.
• the term "overproducing strain” is intended to mean a strain of S. aureus which has been genetically modified to produce at least 10, advantageously at least 20 times more capsular polysaccharide by weight per unit volume in the culture supernatant, than the parent strain from which it originated when the two strains are cultured under the same culture conditions.
• the amount of capsular polysaccharide produced can be determined by the ELISA method as described in Example 1.
• a "genetically modified" strain is a S. aureus strain whose genome has been genetically engineered to increase its capsular polysaccharide production capacity.
• modification of the S. aureus strains may be cited by homologous recombination of the main promoter of the cap ⁇ or cap8 operon by a strong main promoter of another strain such as that the main promoter of the cap1 operon of S. aureus strain M.
• the method of gene insertion by homologous recombination is described in detail in the article by AJ Link et al (J. of Bacteriology, Oct. 1997, p.6228-6237) and its specific application to the production of a strain. Overproducing S.
• aureus is described in detail in the TT article. Luong et al (Infection and Immunity, July 2002, p3389-3395). We can therefore refer to these articles for more details on the operating conditions that can be used.
• This same method can also be used to construct a recombinant strain that overproduces the T5 capsular polysaccharide. It may be necessary for this purpose to adapt certain parameters, the homologous recombination being more or less easy to implement depending on the starting strain.
• an overproducing Reynolds CYL1892 strain in which the main promoter of the cap ⁇ operon was replaced by homologous recombination by the main promoter of the cap1 operon of S.
• aureus strain M could be produced by the method of Luong et al in which the following modifications were made: the homologous sequences were replaced by equivalent sequences homologous to the Reynolds strain, the plasmid pCL52.2 was replaced by the plasmid pCL10 containing a heat-sensitive origin of replication and comprising a cat (chloramphenicol acetyl tranferase) marker conferring resistance to chloramphenicol). Briefly a DNA fragment of about 1 kb containing the capd ORF of the Reynolds strain fused to a fragment of about 250-bp containing the promoter Pcapi is constructed by the overlap PCR technique.
• a DNA fragment of about 1 kb containing the upstream sequence of the cap ⁇ A gene is amplified by PCR from the genome of the Reynolds strain.
• the two fragments are then cloned into the plasmid pCL10 bearing a thermosensitive origin of replication and comprising a cat tag so that the Pcap ⁇ promoter is replaced by the Pcapi promoter.
• the plasmid thus constructed is then electroporated in a strain RN4220 and then introduced by transduction using bacteriophage 52 into the Reynolds strain.
• the sensitivity to the temperature of the replication origin of the plasmid then makes it possible to promote homologous recombination by blocking its replication while applying a selection by chloramphenicol.
• CYL770 strain is meant the overproducing S. aureus strain, obtained from a Becker wild-type strain (CIP103314) in which the capB operon promoter has been replaced by the strong promoter of the cap1 operon.
• S. aureus strain M as described by Luong and Lee (2002 Infect., Mellun 70: 3389-3395).
• strain CYL1892 denotes the overproducing S. aureus strain obtained from a Reynolds strain (CIP 103313) in which the promoter of the cape operon has been replaced by the strong constitutive promoter of the cap operon. 7 of S. aureus strain M, according to the method described by Luong and Lee (2002 Infect.lmmun 70: 3389-3395) including the modifications described above.
• the overproducing strains of S. aureus in particular the CYL770 and CYL1892 strains, have a high genetic stability.
• High genetic stability is understood to mean the property of the strains to retain their capacity for overproduction after at least 25 passages on a culture medium suitable for culturing capsulated S. aureus strains, as measured by the test described in Example 1
• the inventors have shown that the capsular polysaccharide produced by the overproducing strains of S. aureus (hereinafter referred to as rPS) has physico-chemical characteristics different from those of the PS produced by the corresponding wild-type strain (called by the continued sPS).
• PS8 and PS T8 are used interchangeably in the context of the present application to designate a capsular polysaccharide serotype 8.
• PS5 and PS T5 are used interchangeably in the context of this application to designate a serotype 5 capsular polysaccharide.
• the polysaccharide rPS T8 has a mean molecular weight much higher than the corresponding sPS. 250 kDa versus 150 kDa.
• the polysaccharide rPS T5 has a mean molecular weight much greater than the corresponding sPS. 150 kDa versus 50 kDa.
• the inventors have shown that the T5 rPS is mainly released in the culture supernatant.
• rPS exhibit adsoption properties on a plastic surface different from those of sPS.
• Anti-PS8 polyclonal antibodies were generated in the rabbit by repeated injections of different T8 strains (Becker, Wright and CYL770), previously grown in M1 medium.
• the anti-PS8 antibody titers of these sera were evaluated by ELISA using different PS8 (purified from Becker, Wright or CYL770 strains) as adsorption antigen.
• PS8 purified from Becker, Wright or CYL770 strains
• the results showed that anti-PS8 polyclonal serum, generated from the Becker strain, has a high anti-PS8 antibody titer. This title is similar, whether it is evaluated on the PS8 of the Becker strain or on the PS8 of the Wright strain.
• a weak anti-PS8 titre is observed when this one is evaluated with adsorbed PS8 which has been purified starting from the strain CYL770.
• the same results were obtained for the sera generated from the Wright or CYL770 strains
• the inventors evaluated the cross-reactivity of an anti-CYL770 serum against different S. aureus type 8 strains.
• a hyperimmune serum was prepared in the rabbit by injection of whole seeds formulated with the strain CYL770 and adsorbed on chemically autocapsed autologous seeds.
• the results obtained show that the anti-strain serum CYL770 is cross-reactive with Becker and Wright strains with a T8 sPS on their surface.
• the rPS can be produced by a production process comprising the steps of: a) cultivating an overproducing S. aureus strain, in particular a strain CYL1892 or CYL770, b) inactivating the culture thus produced, and c) recovering the T5 or T8 capsular polysaccharide from the culture supernatant.
• Step (a) may be carried out on a solid or liquid culture medium.
• Any culture medium described in the literature as being suitable for culturing a capsulated S aureus strain can be used for this purpose.
• the following media may be mentioned: TSB broth (trypticase soya); defined medium of Poutrel (Poutrel et al., Clin Diagn Lab Immunol, 1995 2 (2): 166-71); Columbia Bouillon. Reference is made thereafter to inductive or non-inductive media.
• the term "inducing medium” means a culture medium which contains an element (for example NaCl or another salt such as CaCl 2 and MgCl 2 ) which acts indirectly or directly on the inductively induced main promoter of the cap locus.
• An inducing medium is thus a medium capable of inducing capsule production in S. aureus strains comprising a principal promoter inductively in the cap locus.
• a non-inducing medium is a culture medium that does not contain such a signal.
• protein of animal origin proteins obtained from matter of animal origin as well as all products derived from it, such as derivatives derived from the chemical treatment of animal proteins.
• the products of the partial or total hydrolysis of these animal proteins such as the peptones, the polypeptides, the peptides or the amino acids deriving therefrom are also meant.
• the culturing step can be carried out by seeding the culture medium with an inoculum, for example with an initial OD 68 O nm of 0.2, and incubating at 37 ° C. for a duration of, for example, approximately 48 to 72 hours. in an atmosphere containing or not CO 2 at a concentration of 5 to 10%
• the volume of culture can vary according to the needs. Volumes of 400ml to 200l or higher volumes for example 301 to 1001 can be used. Preferably a volume ratio of the culture medium to the volume of the culture vessel of 20% (V / V) is maintained to promote oxygenation. For large volumes, oxygenation is controlled and regulated during culture by adjusting the oxygen pressure and appropriate agitation.
• the culturing step may include successive cultures in increasing volumes to increase the biomass and amount of polysaccharide that is produced.
• the culture resulting from step (a) is then subjected to an inactivation step (b).
• the inactivation step may be carried out by treating the culture with a phenol / ethanol mixture (1V / 1V) at a rate of 2% by volume in the end, and then stirring, for example using a magnetic bar. at room temperature for 6 hours to 48 hours depending on the biomass to be treated. inactivation is controlled by a mortality test.
• the rPS contained in the culture supernatant can then be purified by any conventional PS purification technique.
• Various purification techniques have been described in the literature and can be used in the context of the present invention.
• the articles by Lee J.C. et al., 1987; 55: 2191-2197 or Fattom et al Infect. Immun. 1990 ; 58: 2367-2374 the latter describing a process involving release of PSs by lysostaphin, RNAse, DNAse, protease, fractional ethanolic precipitation, ion exchange and molecular sieving treatments.
• preference will be given to a technique giving rise to an rPS having a degree of purity of at least 70%.
• the physico-chemical analyzes and the 500 MHz one-dimensional proton nuclear magnetic resonance analysis show that the T8 and T5 rPS purified from an overproducing strain have a structure conforming to that described in the literature with a purity of at least 70% and a level of O- and N-acetylation greater than 70%. Residuals are present in the final product at less than 5% (w / w) of protein residues, less than 1% (w / w) of residual nucleic acids and less than 5% (w / w) of residual lipoteichoic acid and teichoic acid.
• the amount of capsular polysaccharide obtained can be evaluated by the ELISA technique as described in Example 1. It was thus shown that the strain CYL770 produced in the supernatant a quantity of rPS T8 approximately 80 times higher than that produced by the parent strain Becker. This productivity gain remains stable after successive passages in culture medium. The inventors have also shown that the CYL1892 strain produced about 50 times more T5 rPS, ie 200 ⁇ g / ml on average, than the parent wild-type strain. This productivity gain was essentially observed in the culture supernatant and remained stable after successive passages in culture medium. On the other hand, strain CYL1892 produces 7 times more T5 polysaccharide at the level of the cell pellet than the wild-type parent strain.
• the T5 rPS recovery step may also include an additional step of extracting the T5 rPS from the cells.
• Various extraction techniques have been described in the literature for this purpose and can be used in the context of the present invention. For example, reference can be made to Lee J. C. Infect. Immun. 1993; 67: 1853-1858; Dassy B. et al. Gen. Microbiol. 1991; 137: 1155-1162 for a detailed description of these techniques.
• the PS production method according to the invention can therefore be advantageously used to produce T5 rPS, preferably from a CYL1892 strain, directly from the PS extraction supernatant from the cell pellet.
• the method according to the invention thus makes it possible to simplify the heavy steps of elimination of the numerous cellular contaminants that are for example: DNA, RNA, teichoic acid.
• the subject of the invention is also a capsular polysaccharide rPS, in particular a T5 rPS of the S. aureus strain CYL1892.
• Said polysaccharide is advantageously in isolated or purified form.
• the rPS T5 and T8 according to the invention can be fractionated or depolymerized, for example according to the method described in US Pat. No. 6,045,805, to which reference can be made for a complete description of the operating conditions. In the context of the present invention, the terms “fractionate” and “depolymerize” are used interchangeably. These rPS Fragments are hereinafter called rPSf.
• the T5 and T8 rPS according to the invention are advantageously fractionated or depolymerized until an rPSf is obtained having a mean molecular weight advantageously in a range of from 10 to 120 kDa, in particular in a range from 30 to 70 kDa such as a mean PM rPSf of 5OkDa.
• the subject of the invention is therefore also a capsular polysaccharide rPSf, in particular a rPSf T5, advantageously a T5 rPSf of the strain of S. aureus CYL1892.
• Said polysaccharide rPSf is advantageously in isolated or purified form.
• the invention also relates to the use of the T5 and T8 rPS capsular polysaccharides produced by the overproducing S. aureus strains, advantageously the CYL1892 and CYL770 strains, to produce immunogenic compositions and vaccines for the treatment or prevention of strains of S. aureus T5 and / or T8.
• the capsular polysaccharides rPS induce the production of antibodies that can mediate the destruction of the bacteria that carry them by phagocytes in the presence of complement.
• the invention therefore relates to an immunogenic or vaccine composition
• an immunogenic or vaccine composition comprising a T8 and / or T5 rPS of an overproducing S. aureus strain, advantageously of a CYL770 or CYL1892 strain.
• the invention also relates to the use of a T8 and / or T5 rPS of an overproducing S. aureus strain, advantageously of a strain CYL770 or CYL1892 for the manufacture of an immunogenic composition or for the manufacture of a vaccine composition for the prevention and / or treatment of S. aureus infection.
• said immunogenic or vaccine composition makes it possible to induce a humoral response against strains of S. aureus T8 and / or T5 in the subject to whom it is administered.
• said composition comprises the rPS T8 and T5.
• an immunogenic or vaccine composition is particularly advantageous since it makes it possible to induce, in the subject to whom it is administered, an immune response against S. aureus strains T5 and T8, which represent the vast majority of S. aureus infectious strains.
• the immunogenic or vaccine composition according to the invention can be used to fight infections in infected subjects or to prevent it in risky subjects such as those hospitalized or undergoing surgery.
• the immunogenic or vaccine composition according to the invention is intended for a child under 2 years of age or a subject with a weakened immune defense, such as an elderly person (> 60 years).
• the rPS according to the invention is preferably used in a conjugated form.
• the present invention therefore relates to a rPS-carrier protein conjugate.
• carrier protein is meant a protein that allows for cell collaboration between T and B cells in the induction of the immune response against the capsular polysaccharide and thus improves immunogenicity whether for active immunization or for passive immunization using high titre antisera prepared on volunteers.
• the carrier proteins are preferably non-toxic and non-reactogenic proteins.
• Non-limiting examples of carrier proteins that may be used include recombinant or inactivated bacterial toxins such as Pseudomonas aeruginosa exotoxin A, tetanus, diphtheria or pertussis toxoids, Staphylococcal toxoids or exotoxins, or heat-labile toxin ( LT) or Shiga toxins (ST) from E.
• Bacterial outer membrane proteins may also be used, such as, for example, the outer membrane complex (OMPc), porins, transferrin binding proteins, pneumococcal surface protein A (PsaA). Still other proteins such as bovine serum albumin (BSA), keyhole limpet hemocyanin or purified protein derived tuberculin (PPD) can be used as a carrier protein.
• OMPc outer membrane complex
• PsaA pneumococcal surface protein A
• BSA bovine serum albumin
• PPD purified protein derived tuberculin
• the polysaccharides T5 and T8 rPS are preferably "fractionated” or “depolymerized” before conjugation to the carrier molecule, for example according to the method described in US Pat. No. 6,045,805 to which reference may be made for a description operating conditions to use. Unfractionated rPS can lead to a yield cumulative conjugation and weaker purification and conjugates whose sterilizing filtration may be more difficult to implement because of their larger size.
• the depolymerized or fractionated rPS are named in the rest of the rPSf application.
• the rPS according to the invention can be depolymerized or fractionated until rPSf is obtained having an average molecular weight of 10 to 120 KDa, in particular 30 to 70 KDa, such as 50 kDa.
• the rPS T5 of strain CYL1892 is depolymerized or fractionated until obtaining an rPSf having a mean molecular weight advantageously in the range 30 to 70 kDa, for example 50kDa
• the rPS T8 of the strain CYL770 is depolymerized or fractionated until an rPSf is obtained having a mean molecular weight advantageously in a range of 30 to 70 kDa, for example 5OkDa.
• the polysaccharide is then functionalized before being conjugated to the carrier protein by covalent bonding.
• the functionalization can be carried out according to different methods. For example, activated carboxylate groups of the polysaccharide may be functionalized with adipic acid dihydrazide (ADH) or cystamine, and then the polysaccharide may be conjugated to the carrier protein by a carbodiimide reaction of the partially amidated polysaccharide with a group. carboxylate of the carrier protein.
• Hydroxyl groups of the polysaccharide may also be activated using cyanogen bromide or 1-cyano-4-dimethylaminopyridium tetrafluoroborate, and then the polysaccharide may be functionalized with ADH according to the method described by Kohn et al. (1993 FEBS Lett., 154: 209: 210).
• the functionalized polysaccharide is then bound to the carrier protein, for example recombinant Pseudomonas aeruginosa exoprotein A in which the GLU 553 residue has been deleted, which is referred to herein as rEPA, in the presence of ethyldimethylaminopropyl carbodiimide (EDAC).
• the invention therefore relates to a conjugate comprising PSf T8 and / or T5, advantageously CYL770 and CYL1892 respectively, conjugated to a carrier protein, preferably detoxified ExoA rEPA.
• the invention also relates to a conjugate comprising rPSf T8 and / or T5, advantageously CYL770 and CYL1892 respectively, conjugated to a carrier protein, preferably detoxified ExoA rEPA.
• the invention relates to a mixture comprising the conjugates as defined above, in particular the PSf T8 and T5 conjugates or the rPST ⁇ and T5 conjugates, in which the PSs are conjugated to an identical carrier protein or different, advantageously identical and corresponding to the detoxified ExoA.
• the conjugates according to the invention can be used for the manufacture of an immunogenic or vaccinal composition, in particular a vaccine composition intended for the prevention and / or treatment of S. aureus infection.
• the invention therefore also relates to an immunogenic or vaccine composition comprising said conjugates or their mixture as defined above.
• the compositions according to the invention are useful for inducing a humoral response in vivo, in particular for producing antibodies or antisera against a S. aureus T5 or T8 strain, or for combating infection in infected subjects or preventing it in subjects at risk such as people hospitalized or undergoing surgery.
• the subject of the present invention is therefore also the preparation of said antibodies and antisera, which can be carried out by the conventional techniques for obtaining antisera and polyclonal and monoclonal antibodies well known to those skilled in the art, as well as the antibodies and directed antisera thus produced.
• the immunogenic or vaccine compositions according to the invention may be prepared by any usual method known to those skilled in the art.
• the antigens according to the invention are mixed with a pharmaceutically acceptable excipient.
• pharmaceutically excipient means any excipient, filler, diluent, vehicle, preservative etc, which is conventionally used in the preparation of the compositions administrable to humans.
• these products are selected according to the chosen dosage form and route of administration and according to standard pharmaceutical practices.
• Remington's Pharmaceutical Sciences which is a reference manual in the field.
• compositions according to the invention may further contain an adjuvant.
• an adjuvant Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose.
• suitable adjuvants are aluminum salts such as aluminum hydroxide or aluminum phosphate, DC-Chol and ToII agonists.
• the invention relates to a method of immunizing a subject against S. aureus infection comprising administering to said subject an immunoperatively effective amount of an immunogenic or vaccine composition of the invention. as defined in the description above.
• subject any subject at risk for an infection with S. aureus, such as for example a person hospitalized or having to undergo surgery.
• compositions according to the invention may be administered by any conventional route, usually used in the field of vaccines, such as the parenteral route (intravenous, intramuscular, subcutaneous, etc.).
• parenteral route intravenous, intramuscular, subcutaneous, etc.
• preference will be given to injectable compositions and intramuscular administration.
• Such administration can be performed advantageously at the level of the muscles of the thigh or the arm.
• the compositions according to the present invention may also be administered orally.
• Administration via the nasal, vaginal or rectal mucosa may also be recommended in the context of the present invention.
• Administration may be by administration of a single dose or repeated doses.
• a vaccine dose may be prepared in a volume of 0.1 ml to 2 ml, preferably in a volume of 0.5 ml.
• a Immuno-effective amount is an amount capable of inducing, in a vaccinated subject, a humoral response comprising antibodies capable of opsonizing a strain of S. aureus and facilitating its removal by the phagocytes of the infected individual.
• the composition may be advantageously administered at least 10 to 14 days before the start of the period of increased risk of S. aureus infection.
• Figure 1 represents the amount of capsular polysaccharide type 5 (PS5) measured in the pellet or culture supernatant for the Reynolds strain or the recombinant strain CYL1892.
• Figure 3 represents total anti-PS8 IgG titers induced in mice immunized with T8-rEPA PS conjugates prepared from CYL770 strain or Becker strain.
• the value of the titers is represented by the log of the average of the dilution inverses.
• the individual title of each serum is represented by a symbol.
• the non-specific response measured in preimmune animals (J-1) is also shown.
• FIG. 4 represents the titers of IgGI, IgG2a and IgG3 anti-PS8 subclasses, measured on D35 in mice immunized with T8-rEPA PS conjugates prepared from strain CYL770 or the Becker strain.
• the value of the titers is represented by the log of the average of the dilution inverses.
• the individual title of each serum is represented by a symbol.
• the non-specific response measured in preimmune animals (J-1) is also represented.
• FIGS. 6 (a) and (b) respectively represent the spectra obtained by one-dimensional and 500 MHz proton NMR analysis of the rPS5 polysaccharide purified from the culture supernatant of strain CYL1892 and sPS5 purified from the pellet. cell culture of a Reynolds strain.
• Figures 7 (a) and 7 (b) respectively represent the evaluation and comparison of anti-PS5 IgM and IgG responses induced in mice by the PS5-rEPA and rPS5-rEPA conjugates.
• composition of the culture media used in the following examples is as defined in Table 1 below. Said media were prepared by mixing the constituents in the order indicated in the table. "
• the frozen product (A) comes directly from the original frozen product by amplification on a Columbia medium and contains, as it does, materials of animal origin.
• a second freeze (B) was derived from the original freezing by cloning and repeated passages on media free of animal material.
• a pre-culture medium TSB (trypticase soy broth) was seeded from 100 .mu.l of initial frozen material and placed under stirring for 18 hours at 37 0 C. A 400 ml culture was then inoculated from the preculture. After stirring for 5 hours at 37 ° C., 200 ml of culture are added to 50 ml of glycerol filtered over 0.22 ⁇ m. The distribution of the frozen and carried out under 100 tubes of 100 ⁇ l and 100 tubes of 1 ml then storage at -80 ° C.
• TSB trypticase soy broth
• Cloning steps are performed on non-inductive MO medium. From an aliquot of 100 .mu.l of the original strain CYL1892, isolation is carried out on non-inducing MO medium. From this isolation, two colonies are stitched, taken up with 50 ⁇ l of nuclease-free H 2 O and then seeded again in isolated colonies. After three passages, one colony per box is taken up and then seeded into a sheet (a volume of colony recovery is taken in parallel to perform a PCR control). The web thus obtained is then used for the inoculation of a 5 hour culture of liquid MO medium (50 ml).
• the optical density at 680 nm is measured, then 40 ml of culture are added to 10 ml of coded glycerol and filtered 0.22 ⁇ m.
• the distribution is carried out under 45 x1 ml and stored at -80 ° C.
• Culture and freezing of the frozen B Serial dilutions are carried out in order to eliminate any components of animal origin possibly present in the original frozen.
• the non-inductive MO medium is used during the passages.
• a 100 th dilution is carried out on the pre-freeze B starting before the first passage on agar medium. Serial dilutions (10 "5) are then performed between each passage (passages 2-4). A total of four passages on agar medium were performed with a total dilution factor of 10 '17. The web corresponding to the last dilution carried out is used to inoculate a culture of 400 ml of media MO inductor liquid. the culture is stopped at the end of the exponential phase after 5 hours of culture at 37 ° C with stirring.
• capsular typing by T5 / T8 multiplex PCR amplification is carried out from isolation on Columbia 2% NaCl agar.
• the Reynolds and Becker strains are used as controls for the T5 and T8 amplifications.
• a cap1 genetic stability PCR test using the oligonucleotides Ppai Ncol and Ppair (described by Luong et al in Infect Immun 2002, 70 (7): 3389-95) is also carried out: isolation is carried out on 2% NaCl Columbia agar. and incubated for 24 hours at 37 ° C. A colony is then recovered to make a second passage on Columbia 2% NaCl agar. Four successive passages are thus made. L 1 DNA of the colonies was extracted by PCR to verify the presence of the promoter Cpl CYL770 The strain is used as a control for amplification of the promoter Cpl.
• a pre-culture in the liquid MO medium is seeded from the frozen test.
• a culture of 400 ml in M1 inducing medium is then seeded and stirred at 37 ° C for 72 hours.
• a volume of 50 ml of culture is taken and centrifuged 30 minutes at 3500 rpm at + 4 ° C.
• the capsular polysaccharides are then extracted and assayed by ELISA.
• Successive cultures in M1 medium are carried out so as to generate a number of generations close to what could be obtained in a 1000 liter fermenter.
• the suspensions are centrifuged for 30 minutes at 3500 rpm at + 4 ° C., the culture supernatants are separated and stored at + 40 ° C.
• the pellets are extracted by means of two successive cycles of autoclave. Each cycle consists of a recovery of the pellets in 5 ml of PBS followed by a 60 minute autoclave cycle at 121 ° C.
• a mortality test on the autoclaved pellets is carried out by inoculation on Columbia 2% NaCl agar.
• the autoclaved pellets are then centrifuged for 30 minutes at 25,000 ⁇ g at + 40 ° C. and the extract (supernatant) recovered and then stored at + 4 ° C.
• the pellet is then taken up again in 5 ml of PBS and a second autoclave cycle is started. At the end of the second cycle, the two extracts are grouped together.
• the extracts and culture supernatants are treated with proteinase K (50 ⁇ g / ml) for 1 hour at 37 ° C. and then proteinase K is inactivated by incubation for 30 minutes at 80 ° C. A mortality test is then performed on the treated culture supernatants.
• Proteinase K treated samples are assayed in sandwich ELISA.
• a double Titration ensures the reliability of the data.
• the principle is to capture the antigen (PS5) between two antibodies to quantify it. In order to avoid any interference and to obtain the most accurate measurement possible, one of the two antibodies must be perfectly specific (anti-PS5 monoclonal antibody used for adsorption). An adsorbed rabbit polyclonal serum is then used as the detection antibody. A secondary antibody conjugated to peroxidase will allow the revelation.
• a standard curve is performed with purified PS5, used at different concentrations to quantify the antigen.
• a control is also introduced, it is purified PS5 whose concentration is known and established by physicochemical measurement at 78 ⁇ g / ml.
• a chromogenic substrate tetramethyl benzidine (TMB) is added. This is degraded by peroxidase into a blue colored product.
• An acid is added after 15 minutes of incubation at 20 ° C. and in the dark, which causes the reaction to stop and transforms the blue colored product into yellow.
• the optical density is measured at 450 nm, it is proportional to the amount of antibody fixed.
• PCR Profile The genetic stability of the pre-congelate was monitored during the various cloning steps. The presence of a specific amplification of cap ⁇ at around 500 bp and of the cap1 promoter at 200 bp is noted. The ten colonies tested are derived from the pre-congelate B used for the seeding of the culture for the production of frozen B. The genetic stability of the cap ⁇ and cap1 promoter loci is observed.
• the recombinant strain CYL1892 produces on average 3.5 times more PS5 in the supernatant than in the pellet.
• the total amount thus produced represents approximately 10 times more PS5 compared to the Reynolds wild-type strain (300 ⁇ g / ml and 30 ⁇ g / ml, respectively) after culture in M1 liquid inducing medium.
• the difference is very significant in supernatants.
• Approximately 50-fold higher PS5 production is observed for strain CYL1892 relative to the Reynolds wild-type strain.
• the difference is much less important with regard to the pellets. Indeed, we observe a PS5 productivity difference of a factor 2 for the strain CYL1982 relative to the Reynolds strain. It is found that the CYL1892 B frozen product produces an equivalent amount of PS5 with respect to the frozen A.
• T8 and T5 rPS isolated and purified from the CYL770 and CYL1892 strains respectively were characterized by the analyzes described below.
• rPS5 and rPS8 no unidentified or identified glycosidic impurity (such as peptidoglycan, teichoic acid, lipoteichoic acid, and antigen 336), likely to come from S. aureus, was detected by NMR analysis.
• the NMR spectra of rPS8 and rPS5 purified from strains CYL770 and CYL1892 respectively are given in FIGS. 5 and 6.
• High performance steric exclusion chromatography was performed using as in-line detectors a light scattering detector, a viscometer and a refractometer. This analysis evaluated the average molecular weights of rPS8 and rPS5 at 250 kDa and 150 kDa, respectively. These molecular weight values are higher than those of capsular polysaccharides purified from the S. aureus Becker (type 8) or Reynolds (type 5) strain cultured with the same medium and under the same culture conditions and purified from the cell pellet (average molecular weight estimated at 150 kDa and 50 kDa, respectively).
• a culture of the type 8 recombinant strain CYL770 was carried out in M1 inducing medium. Germs derived from this culture have been formulated for use in an immunization protocol in rabbits according to the principle described in the literature (Karakawa et al., 1985 J Clin Microbiol 22: 445-447).
• the hyperimmune serum obtained contains antibodies specific for type 8 capsular polysaccharides, but also antibodies directed against other epitopes that may be common between the different strains. This serum was therefore adsorbed several times on pellets at 10 11 CFU of the autologous strain rendered chemically acapsulated so as to eliminate nonspecific antibodies from the capsular polysaccharide type 8. The reagent thus obtained was then evaluated by agglutination technique.
• the culture is then centrifuged, the pellet is taken up in 20 ml of PBS (stock solution) and the OD ⁇ sonm is measured. A volume of stock solution is taken to perform the count before formulation.
• stock solution CYL770, dilutions of reason 10 are made in 24-well plate of 10 '1 to 10 ' 9 .
• Six drops of 25 ⁇ l of each dilution are seeded on Columbia 2% NaCl agar and incubated for 18 to 20 h at 37 ° C.
• a mortality test is carried out by amplification of 1 ml of formalin suspension inoculated in 9 ml of TSB and placed with x100 stirring at 37 ° C. for 18 to 20 hours, then inoculation of a mortality test on Columbia agar.
• the formulated germs are adjusted and aliquoted under 4ml in 5mL sterile flasks.
• the fit of the formulated seed suspensions is made with respect to the OD 680 nm equivalent to the required seed concentrations:
• a preculture of 50ml of Columbia medium is seeded and incubated at 37 ° C with stirring for 18h.
• a 400ml culture of Columbia medium is seeded at an initial OD beta of 0.2 and incubated at 37 ° C. with stirring for 24 hours.
• the culture is then centrifuged at 3500rpm for 30min at + 4 ° C and the supernatant is removed.
• the pellets are then taken up and pooled with 30 ml of PBS and an OD measurement is made to determine the distribution volume per tube so as to have a total of 10 11 CFU. After centrifugation at 3500 rpm for 30 min at +4 ° C. and removal of the supernatant, the dry pellets are stored at -20 ° C. until use.
• Serum Adsorption on Chemically Acapsulated Germs Two ml of anti-CYL770 polyclonal serum were used for the adsorption protocol. A bacterial pellet of 10 11 CFU makes it possible to carry out an adsorption cycle for 2 ml of serum. A total of 5 adsorption cycles were carried out, each cycle being composed as follows: deposit 2ml of anti-CYL770 decomplemented serum on a pellet of chemically-acapsulated seeds, resuspend gently, place on a rotator for tubes at + 4 ° C on the day or at night, centrifuge at 3500rpm for 30min at + 4 ° C, recover the serum adsorbed, deposit the serum on a new pellet of chemically-acapsulated germs and resume the protocol.
• the adsorbed serum is recovered and filtered through a 0.22 ⁇ m Millipore membrane and then stored at + 4 ° C. until evaluated by slide agglutination test.
• Serum adsorbed agglutination test The agglutination test on the slide of polyclonal anti-CYL770 serum adsorbed on chemically-acapsulated seeds was carried out on trypsinized seeds derived from cultures on different solid and liquid media of S. aureus type 8 strains ( Becker, Wright and CYL770). Non-PS inducing solid culture of the Reynolds-type S. aureus strain was also tested as a control.
• D ⁇ 680nm 5.
• liquid cultures of 50 ml in TSB medium and in M1 medium were made from solid precultures in TSB and MO inducing agar medium, respectively.
• the solid cultures were seeded directly from a frozen as previously described. Layers of the solid cultures were resuspended in PBS and were used to inoculate the liquid cultures with an initial OD beta of 0.2.
• a liquid culture of 50 ml in M1 medium was performed.
• a preculture of 10 ml in MO medium liquid inducer was directly seeded from a freezing of 1ml.
• the culture in M1 medium was seeded at an initial OD 680 nm of 0.2 from the liquid preculture.
• the liquid cultures were incubated for 24 hours at 37 ° C. with shaking.
• trypsin so as to eliminate any troublesome trace of protein A.
• 100 ⁇ l of trypsin to 50 mg / ml were added and the mixture incubated for 1 h at 37 ° C. on a rotator and then centrifuged at 3500 rpm for 30 min at + 40 ° C.
• the suspensions are stored at +4 ° C. until use.
• a seed control was first made with 50 .mu.l of suspension of germs on which 5 .mu.l of PBS were deposited. The control is validated in the absence of agglutination after 5 minutes of contact.
• a volume of 5 ⁇ l of anti-CYL770 polyclonal serum adsorbed on chemically decapsulated seeds was used for the agglutination tests.
• the blade was manually shaken in a circular motion over a Kahn mirror (mirror concave). This step was timed from the addition of the serum in the first well.
• the strain CYL770 is cultured in M1 medium for 48 hours, it is then fixed with formalin. Direct mortality as well as mortality after amplification is verified. The suspension is then adjusted to the required concentrations (Table 2) and divided into sterile vials. The vials are then crimped and used for immunization according to the protocol described in the literature (Karakawa et al., 1985 J Clin Microbiol 22: 445-447).
• the serum is adsorbed on chemically-activated autologous seeds and then evaluated against type 8 strains in slide agglutination (Table 3).
• MO M1 TSB M1 MO M1 TSB Agar Agar Agar Agar Agar Inducer Inducer
• the reading of the agglutination test was carried out as follows: ++++ total agglutination observed in 0 to 30 sec +++ total agglutination observed in 30 sec at 1 min ++ total agglutination observed in 1 to 3 min
• the anti-CYL770 serum adsorbed agglutin all the more rapidly the germs when they carry the type 8 capsule.
• the same agglutination profile is observed with the anti-PS8 monoclonal antibody (Mab-PS8).
• the adsorbed anti-CYL770 serum therefore, showed no difference in recognition of the three strains tested when they were placed under capsule inducing conditions. Since agglutination is related to the presence of capsular antigens, no difference between type 8 capsular polysaccharides produced by these different strains is therefore worthy of note.
• anti-PS8 polyclonal antibodies were generated by the immunization of rabbits with inactivated whole seeds of the strain CYL770 grown in M1 medium, taking inspiration from the protocol previously described by Karakawa and collaborators
• Blood collection from preimmune animals Five to 10 ml of blood are collected in Vacutainer TM tubes prior to the first immunization (D-1) for each rabbit, locally anesthetized with Xylocaine, in the medial artery of the patient. hear. Samples are left to exude during 3h to 4h at room temperature (20-22 0 C) and then centrifuged at 4 ° C for 20 min at 1500 g
• Exsanguination Blood Collection All animals are removed by cardiac puncture under general anesthesia, 28 days after the first injection. Between 50ml and 80ml of blood are collected per animal and collected in sterile Falcon TM 50ml tubes.
• the polyclonal sera of rabbits are evaluated for their specificity with respect to PS8 by an ELISA test.
• 96-well ELISA plates flat-bottom microplates, Immunosorp Microwell, Nunc, Roskilde, Denmark
• the plates are washed 4 300 ⁇ l / well of PBS / 0.05% Tween 20 (PBS-Tween) using the Titertek M96V Washer automatic plate washer then saturated for 1 hour at 37 ° C. with 250 ⁇ l / well of PBS-Tween / 1 % BSA.
• the plates are then washed 4 times according to the method described above. 100 ⁇ l per well of each of the sera to be tested at different concentrations are deposited in the plate, the successive dilutions of reason three are carried out within the plate itself (dilution in column) using a multichannel pipette in PBS -Tween 1% BSA. The plates are incubated for 1 h 30 at 37 ° C. and then washed 4 times with PBS-Tween. Rabbit anti-IgG conjugate conjugated to HRP (Southern Biochemicals)
• the anti-PS8 specific titres of the polyclonal antibodies correspond to the inverse of the arithmetic mean of the dilutions observed for two wells at 450 nm, relative to a reference serum.
• rPS8 antigen purified from strain CYL770 is depolymerized according to the principle described in US Pat. No. 6,045,805.
• the rPS8 antigen is prepared at 2 mg / ml.
• To 20 ml of this solution at room temperature are added 110 ⁇ l of 200 mM ascorbic acid, 11 ⁇ l of 20 mM CuSO4 and 11 ⁇ l of 20 mM FeSO4.
• the reaction is continued with stirring for 80 min at room temperature.
• the depolymerization reaction is stopped by adding 2 ml of a 2 M TRIS solution at pH 7.0.
• the depolymerization reagents are removed by dialysis against water and then the depolymerized rPS8 is lyophilized.
• the average molecular weight of the depolymerized rPS8 was estimated to be close to 50 kDa by HPSEC / LS / RI / Visc analysis.
• antigen depolymerized for polysaccharide purified from strain CYL770
• ADH adipic acid dihydrazide
• the pH is adjusted to 4.9 and ethyldimethylaminopropylcarbodimide (EDAC) is added to a final concentration of 25 mM.
• EDAC ethyldimethylaminopropylcarbodimide
• the pH is constantly adjusted to a value of 4.9 by addition of a dilute solution of HCl.
• the reaction is stopped by neutralizing the pH.
• the activated antigen is then dialyzed against 500 M NaCl and then against water.
• the activated antigen and dialysis is then lyophilized.
• the percentage of functionalization was estimated at 3.7 and 4.3 (w / w) for the polysaccharide purified from the Becker strain and for the polysaccharide purified and depolymerized from the strain CYL770, respectively.
• Conjugation of the antigen A 10 ml solution containing 20 mg of the activated antigen (and depolymerized for the antigen purified from the CYL770 strain) and 10 or 20 mg of the carrier protein (recombinant exoprotein A ⁇ 553 ie rEPA) in 100 mM NaCl and 50 mM EDAC was prepared.
• the pH is constantly adjusted to a value of 5.6 by adding a dilute solution of HCl.
• the pH is neutralized.
• the conjugated antigen is then dialyzed against 200 mM NaCl and then purified by size exclusion chromatography on a Sepharose CI-4B column equilibrated with 200 mM NaCl in a 10 mM phosphate buffer pH 7.2. Fractions which contained conjugates (as detected by optical absorption at 210 nm and 280 nm) and which are mainly eluted with the dead volume of the column, were pooled.
• the amounts of conjugated polysaccharide and protein were estimated as a weight ratio between the polysaccharide (determined by O-acetyl quantification according to the Hestrin method (Hestrin S. 1949.J.Biol.Chem.180: 249). -261) and the protein (determined by protein quantification according to the method of Bradford (Anal Biochem, 1976 72, 248-254).)
• the level of unconjugated antigen and free carrier protein was determined by capillary electrophoresis. Conjugate size was estimated by HPSEC / LS / RI / Visc.
• the rPS5 antigen is prepared at 2 mg / ml. To 20 ml of this solution at room temperature are added 55 ⁇ l of 200 mM ascorbic acid, 5.5 ⁇ l of 20 mM CuSO4 and 5.5 ⁇ l of 20 mM FeSO4. The reaction is continued with stirring for 80 min at room temperature. The depolymerization reaction is stopped by adding 2 ml of a 2 M TRIS solution at pH 7.0. The depolymerization reagents are removed by dialysis against water and then the depolymerized rPS5 is lyophilized. The average molecular weight of the depolymerized rPS5 was estimated to be close to 50 kDa by HPSEC / LS / RI / Visc analysis. Activation of the antigen
• antigen depolymerized for polysaccharide purified from strain CYL1892
• ADH adipic acid dihydrazide
• the pH is adjusted to 4.9 and ethyldimethylaminopropylcarbodimide (EDAC) is added to a final concentration of 10 mM.
• EDAC ethyldimethylaminopropylcarbodimide
• the pH is constantly adjusted to a value of 4.9 by addition of a dilute solution of HCl.
• the reaction is stopped by neutralizing the pH.
• the activated antigen is then dialyzed against 500 M NaCl and then against water.
• the activated antigen and dialysis is then lyophilized.
• the percentage of functionalization was estimated at 1, 25 and 0.9 (w / w) for the polysaccharide purified from the Reynolds strain and for the polysaccharide purified and depolymerized from the strain CYL1892, respectively. Conjugation of the antigen
• a 10 ml solution containing 20 mg of the activated antigen (and depolymerized for the purified antigen of CYL1892) and 40 mg of the carrier protein (recombinant exoprotein A, rEPA) in 100 mM NaCl and 50 mM EDAC was prepared.
• the pH is constantly adjusted to a value of 5.6 by adding a dilute solution of HCl.
• the pH is neutralized.
• the conjugated antigen is then dialyzed against 200 mM NaCl and then purified by size exclusion chromatography on a Sepharose CI-4B column equilibrated with 200 mM NaCl in a 10 mM phosphate buffer pH 7.2. Fractions which contained conjugates (as detected by optical absorption at 210 nm and 280 nm) and which are mainly eluted with the dead volume of the column, were pooled.
• conjugated antigen After purification, the amounts of conjugated polysaccharide and protein were estimated as a weight ratio between the polysaccharide (determined by O-acetyl quantification according to the method of Hestrin (Hestrin S. 1949 J. Chem. .180: 249-261) and the protein (determined by quantification of protein according to the Bradford method (Anal Biochem, 1976 72, 248-254)).
• the level of unconjugated antigen and free carrier protein was determined by capillary electrophoresis.
• the size of the conjugate was estimated by
• OF1 non-consanguineous female mice (ESD - Charles River Laboratories, St. Germain-sur-l'ArbresIe, France) of 20-22 g are injected subcutaneously into the scapular girdle, at OJ (sensitization) then 3 weeks after the first injection (J21) with different concentrations of PS T8-rEPA conjugates generated with PS8 from strain CYL770 cultivated in M1 medium. The concentrations and volumes used for each injection are described below.
• Table 7 Description of the necessary groups (4 batches of conjugates) for a comparison study of the immunogenicity induced by type 8 conjugates from the wild type Becker strain or the CYL770 strain.
• Prot / PS represents the final ratio after purification. It indicates the proportion of recombinant protein relative to the proportion of PS8 polysaccharide purified by weight / weight.
• mice are anesthetized by intraperitoneal injection of 0.2 ml / mouse of a mixture of 8 mg / ml of Ketamine and 1.6 mg / ml of Xylazine.
• the blood samples are allowed to coagulate and exude for 3H at room temperature (20-22 0 C) and then centrifuged at 4 ° C for 3 min at 6000 g and then transfer into conical tubes of 1 ml Nunc and the sera are stored at -20 0 C until use. Analyzes of the antibody responses
• the anti-PS8 humoral response (anti-PS8 IgG and IgM titers at OJ, J21 and J35, and IgGI, IgG2a and IgG3 anti-PS8 titers at OJ and J35 for the determined immunization dose within the test) is evaluated by an ELISA test using PS8 characterized, purified from T8 Becker strain as antigen for adsorption ("coating") at a concentration of 1, 5 ⁇ g / ml.
• 96-well ELISA plates (M129B Dynex) are incubated overnight at room temperature (20-22 ° C.) in the presence of 1 ⁇ g / ml of purified PS8 starting from the Becker strain in 1X PBS pH 7.2 (100 ⁇ l). / well).
• the plates are washed 4 times with 300 ⁇ l / well of PBS / 0.05% Tween 20 (PBS-Tween) using the automatic plate washer Titertek M96V Washer then saturated for 1 h at 37 ° C with 250 ⁇ l / well of PBS-Tween / 1% BSA.
• the plates are washed 4 times according to the method described above.
• the anti-PS8 titers are calculated using the CodUnit software, for OD values ranging from 0.2 to 3, relative to the reference curve given by the standard serum that is present on each of the plates.
• the titres of each serum are expressed in log- ⁇ 0 .
• the response is low for the 4 groups of conjugates (between 2.5 Log and 2.8 Log) but nevertheless significant in comparison with the responses obtained with the groups immunized with the non-conjugated PS.
• IgG3 a more marked IgG3 response is observed for the 4 batches of conjugates compared to the IgG2a response. No significant difference is observed between the different conjugates.
• the groups that received the unconjugated PS do not have an anti-PS8 lgG3 response
• PS T5-rEPA conjugates were generated using purified PS5 from the Reynolds strain or purified rPS5 from the Staphylococcus aureus recombinant strain CYL1892.
• each conjugate was injected at an optimal dose of immunization (previously defined in dose-effect studies) of 2.5 ⁇ g / mouse / injection).
• OF1 non-consanguineous female mice (ESD-Charles River Laboratories, St Germain-sur-France, France) with a weight of 20-22 g are injected subcutaneously into the scapular girdle at 0 (sensitization) then again at D21, 3 weeks after this first injection with 2.5 ⁇ g / mouse / injection of PS T5-rEPA conjugates generated from purified PS5 from of the Reynolds strain cultivated in M1 medium or from purified rPS5f from strain CYL1892.
• Table 9 Descriptions of the groups required to compare the immunogenicity induced by type 5 conjugates whose PS originate from the wild Reynolds strain or from the recombinant strain CYL1892
• mice are anesthetized by intraperitoneal injection of 0.2 ml / mouse of a mixture of 8 mg / ml of Ketamine (Imalgene) and 1.6 mg / ml of Xylazine (Roumpun).
• the blood samples are allowed to coagulate and exude for 3 h at 20 ° C-22 ° C, centrifuged for 3 min at 6000 g and then transferred into 1 ml Nunc conical tubes.
• the sera are stored at -2O 0 C until they are used.
• anti-PS5 humoral response (anti-PS5 IgM and IgG titers at OJ, J21 and J35) is evaluated by a robotic ELISA using sPS5 purified from the Reynolds type 5 strain as an antigen for adsorption ("coating"). At a concentration of 1 ⁇ g / ml.
• the sPS5 purified from the Reynoldsa strain was chosen for adsorption to evaluate whether the rPS5fT5-rEPA conjugates (origin CYL1892) are capable of inducing an antibody response capable of recognizing specifically purified sPS5 from a strain.
• rPS5fT5-rEPA conjugates oil CYL1892
• All sera specific for PS5 are analyzed by a robotic ELISA (Zymark robot) according to the procedure described below.
• 96-well ELISA plates (M129B Dynex) are incubated overnight at room temperature (20-22 ° C.) in the presence of 1 ⁇ g / ml of purified sPS5. from the Reynolds strain in 1X PBS pH 7.2 (100 ⁇ l / well).
• the plates are washed 4 times with 300 ⁇ l / well of PBS / 0.05% Tween 20 (PBS-Tween) using the automatic plate washer (Titertek M96V Washer) and then saturated for 1 hour at 37 ° C. with 250 ⁇ l. / well of PBS-Tween / 1% BSA.
• the plates are washed 4 times according to the method described above. 100- ⁇ l per well of each of the sera are deposited in the plate.
• the enzymatic reaction due to peroxidase is stopped by the addition of 100 .mu.l of 1N HCl per well.
• the optical density (OD) of each of the wells is measured from 450 nm to 630 nm using an automatic plate reader (Labsystem).
• the background noise (average value over 4 white wells) is subtracted from the measured OD values.
• the anti-PS5 titers are calculated using the CodUnit software, for OD values ranging from 0.2 to 3, relative to the reference curve given by the standard serum that is present on each of the plates.
• IgM induced by the rPS5-rEPA conjugates are capable of specifically recognizing purified PS5 from a type 5 wild-type strain such as the Reynolds strain.
• rPS5-rEPA conjugates show immunogenicity similar to that of the PS5-rEPA conjugates.
• IgG induced by rPS5-rEPA conjugates are capable of specifically recognizing PS5 purified from a type 5 wild-type strain such as Reynolds strain, in the same way as specific IgGs induced by PS5 conjugates. -rEPA.
• the anti-PS5 IgM and IgG responses induced in mice with the rPS5-rEPA conjugates (batch 14 and 15) are very similar to those induced by the PS5-rEPA conjugates (batch 11 and 7) after the prime and after the boost.
• rPS5-rEPA conjugates are capable of inducing high levels of anti-PS5 antibodies that strongly recognize purified PS5 from a wild-type strain such as the Reynolds strain of S. aureus and having an affinity for this antigen similar to that observed for anti-PS5 antibodies induced by PS5-rEPA conjugates.

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