EP2004223A1 - Type 5 and type 8 capsular polysaccharides of superproducing staphylococcus aureus strains - Google Patents

Abstract

The invention concerns type 5 and type 8 capsular polysaccharides produced by superproducing S.aureus strains, as well as immunogenic compositions and vaccines comprising said capsular polysaccharides.

Description

Type 5 and Type 8 capsular polysaccharides of overproducing strains of Staphylococcus aureus

The invention relates to type 5 and type capsular polysaccharides

8 produced by the overproducing S. aureus strains, in particular by the CYL1892 and CYL770 strains, as well as their use for immunization against infection with S. aureus serotype 5 and / or 8 strain.

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. These 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.

The discovery of serologically distinct capsular polysaccharides on the surface of S. aureus clinical isolates led to study their role in the virulence of these strains. Serotyping of S. aureus strains has shown that serotypes 5 and 8 are responsible for the vast majority of human infections.

The development of a vaccine against S. aureus infection is a major issue because of the seriousness of the complications and the ever-increasing emergence of antibiotic-resistant strains. The development of a vaccine protecting against infections with S. aureus strains of serotypes 5 and 8 (T5 and T8) would protect an individual from infection by 85 to 90% of strains of S. aureus.

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. 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 comprising a T8 and / or T5 capsular polysaccharide of a T8 and / or T5 supramatronic S. aureus strain, respectively. According to a particular embodiment, said composition comprises a T5 capsular polysaccharide of a strain of S. aureus CYL1892.

According to another embodiment, said composition comprises a capsular polysaccharide T8 of a strain of S. aureus CYL770.

According to a particular embodiment, 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.

According to a particular embodiment, 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. According to a particular embodiment, said conjugate comprises a capsular polysaccharide T8 of strain CYL770.

According to another embodiment, said conjugate comprises a T5 capsular polysaccharide of strain CYL1892.

According to a particular embodiment, 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.

According to a particular embodiment, said conjugates comprise a fractionated capsular polysaccharide. According to a particular embodiment, the carrier molecule of the conjugate according to the present invention is exotoxin A of Pseudomonas aeruginosa.

In another aspect, 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. According to one embodiment, said PS is split.

According to a particular embodiment, said conjugate comprises a capsular polysaccharide of the S. aureus strain CYL1892.

According to another embodiment, said conjugate comprises a capsular polysaccharide of the S. aureus CYL770 strain.

Said PS are preferably fractionated before being conjugated. In another aspect, the present invention relates to a type 5 capsular polysaccharide of S. aureus strain CYL1892. This polysaccharide is advantageously in isolated or purified form.

In another aspect, 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.

In another aspect, 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.

According to a particular embodiment of the method according to the invention, the overproducing strain is the CYL1892 strain.

The invention will be described in more detail in the description which follows with reference to FIGS. 1 to 6. The term "S. aureus strain (s)" is understood to mean any S.aureus strain expressing a capsule or microcapsule comprising a T5 and / or T8 polysaccharide. By way of non-limiting example, mention may be made of the Newman (T5), Reynolds (T5), Lowenstein (T5), Becker (T8) and Wright (T8) strains. The strains advantageously used in the context of the present invention are the Reynolds and Becker strains.

In the context of the present invention, 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. As an illustrative example of an appropriate genetic modification, modification of the S. aureus strains may be cited by homologous recombination of the main promoter of the capδ 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. By way of example, an overproducing Reynolds CYL1892 strain in which the main promoter of the capδ 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 capδA 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 Pcapδ 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.

By "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).

The term "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 inventors have also demonstrated that 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 On the other hand, 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). The "PS8" and "PS T8" are used interchangeably in the context of the present application to designate a capsular polysaccharide serotype 8. Similarly, the terms "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. On the other hand, unlike the SPS T5 which remains linked to the bacterial wall, the inventors have shown that the T5 rPS is mainly released in the culture supernatant.

The inventors have also observed that 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. 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. On the other hand, 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.

To verify that the weak recognition of the PS8 of CYL770 by the different will be due to a lower capacity of this rPS8 to be fixed on the treated plastic of an ELISA plate, and not to a difference of recognition of the PS8 itself, the three sera were titrated in ELISA on rPS8 of CYL770 conjugated to rEPA, the presence of the carrier protein to greatly enhance the adsorption. Under these adsorption conditions, the anti-PS8 polyclonal sera generated from the 3 different T8 strains, strongly recognize the rPS8 of CYL770, with comparable antibody titers between them and similar to those obtained on PS8 purified from Becker or Wright strains.

In spite of these important differences, which could potentially result in different immunogenic and antigenic properties, the inventors have demonstrated by characterization and immunogenicity experiments that the rPSs retain, on the one hand, their characteristic primary structure as described by C. Jones (Carbohydr Res., 2005, 340, 1097-1106) and retained the immunogenicity and antigenicity of sPS, on the other hand.

In particular, the inventors evaluated the cross-reactivity of an anti-CYL770 serum against different S. aureus type 8 strains. For this purpose, 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. These results therefore demonstrate that it is possible to use the overproducing strain CYL770 to produce large amounts of a T8 capsular polysaccharide for use in an immunization method.

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. By way of non-limiting example, 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. In contrast, a non-inducing medium is a culture medium that does not contain such a signal.

In the context of the present invention, preference will be given to a medium lacking protein of animal origin.

By "protein of animal origin" is meant 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%

(for a strain T5 without CO ₂ and in the case of the use of a strain T8 with CO ₂ ). 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.

As needed, 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. To do this, 100 microliters of the treated culture are spread on a Columbia 2% NaCl agar and in parallel 100 microliters of the treated culture are seeded in trypcase soy broth supplemented with 1/30 ^th of horse serum. The cultures are incubated for 48 hours at 37 ⁰ C ₁ bottle caps broth remaining unscrewed. The absence of viable bacteria results in no growth after 24 hours of incubation at 37 ° C. Once the mortality of the culture is established, the capsular polysaccharide is recovered. In the case of overproducing strains of S. aureus, in particular CYL770 and CYL1892, most of the T8 and T5 rPS are released into the culture supernatant. The rPS can be isolated directly from it. To do this, at the end of the inactivation step, the medium is centrifuged and the cell pellet is removed.

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. For example, 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. In the context of the present invention, 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 μg / 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.

Since part of the T5 rPS remains bound to the bacterial wall, 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.

According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, the invention therefore relates to 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. In particular, 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.

According to a particular embodiment, said composition comprises the rPS T8 and T5. Such 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. When 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.

According to another embodiment, the present invention therefore relates to a rPS-carrier protein conjugate.

By "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. coli. 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.

Because of their high molecular weight, 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. By way of illustration, 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, and 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). Size exclusion chromatography can then be used to separate the conjugates from the unreacted polysaccharides. The functionalization and conjugation activation steps do not alter the chemical structure of the repeating units of the polysaccharide. According to one aspect, 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. In another aspect, 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.

According to a particular embodiment, the invention relates to a mixture comprising the conjugates as defined above, in particular the PSf T8 and T5 conjugates or the rPSTδ 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. Usually, the antigens according to the invention are mixed with a pharmaceutically acceptable excipient. By "pharmaceutically excipient "acceptable" means any excipient, filler, diluent, vehicle, preservative etc, which is conventionally used in the preparation of the compositions administrable to humans. In general, these products are selected according to the chosen dosage form and route of administration and according to standard pharmaceutical practices. One can refer for example to Remington's Pharmaceutical Sciences, which is a reference manual in the field.

The compositions according to the invention may further contain an adjuvant. Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose. Examples of suitable adjuvants are aluminum salts such as aluminum hydroxide or aluminum phosphate, DC-Chol and ToII agonists.

In yet another aspect, 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.

By "subject" is meant any subject at risk for an infection with S. aureus, such as for example a person hospitalized or having to undergo surgery.

The 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.). In the context of the present invention, 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.

The figures and examples below illustrate the invention without limiting its scope.

FIGURES

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 2 shows the anti-PS8 IgM titers induced in mice immunized with T8-rEPA PS conjugates prepared from CYL770 strain or Becker strain. The value of the titers (histogram) is represented by the log of the average of the dilution inverses. For each group, the individual title of each serum (n = 10) is represented by a symbol. The non-specific response measured in preimmune animals (J-1) is also shown.

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 (histogram) is represented by the log of the average of the dilution inverses. For each group, the individual title of each serum (n = 10) 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 (histogram) is represented by the log of the average of the dilution inverses. For each group, the individual title of each serum (n = 10) is represented by a symbol. The non-specific response measured in preimmune animals (J-1) is also represented.

FIGS. 5 (a) and (b) respectively represent the spectra obtained by one-dimensional 500 MHz proton NMR analysis of purified polysaccharide rPS8 from a supernatant of strain CYL770 and sPS8 purified from the pellet. cell culture of the Becker strain. These NMR analyzes were performed using a Bruker DRX 500 spectrometer and a probe: BB-1 HD XYZ GRD 5mm. The samples were taken up in D ₂ O at a concentration of 1.2 mg / ml. The analysis was carried out under the following conditions: analysis temperature: 343 ° K (70 ^° C.); relaxation time: 2s; scanning number = 512. For each PS8, the spectra were made on the native (O-acetylated) form and on the de-O-acetylated form obtained by alkaline treatment as described by C. Jones (C. Jones. Res 2005, 340, 1097-1106). 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. These NMR analyzes were carried out using a Bruker DRX 500 spectrometer and a probe: BB-1H-D XYZ GRD 5mm. The samples were taken up in D ₂ O at a concentration of 1.2 mg / ml. The analysis was carried out under the following conditions: analysis temperature: 343 ° K (70 ^° C.); relaxation time: 2s; scan number = 512.

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.

EXAMPLES

The 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. "

Example 1 Characterization of the strain CYL1892 type 5

A frozen strain of strain CYL1892 was provided by Dr. Chia Y. Lee (Medical Center of the University of Arkansas, USA). This is the original frozen. Preparing the congelats

Two types of congelates have been prepared. 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 μm. The distribution of the frozen and carried out under 100 tubes of 100 μl and 100 tubes of 1 ml then storage at -80 ^° C.

Preparation of the freeze of seed lots A and B Preparation of pre-congelates

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 μl 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). At the end of the culture, 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 μm. 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. measurements of DO ₆ are 80nm The culture is then supplemented with glycerol at 20% final and then divided into 6 Schott flasks kept in the ice until the final distribution in tubes.The distribution is carried out under 1 ml in Nunc tubes. and also in 5 ml in Falcon 15 ml tube The ampoules are then placed at -80 ^° C.

Checks made Viability

Numbering on each distribution vial is performed at the time of distribution of the B-freeze before freezing according to the protocol:

- dilution in PBS of the freeze in series of 10 in 10 until the dilution 10 ^{~ 9} ,

deposit of 6 drops of 25 μl of dilutions 10 ^'3 to 10 ^"9 on 24x24 Nunc plates of Columbia 2% NaCl agar, incubation at 37 ^° C. for 18 hours,

- Colony count in the most readable dilution range and averaging on distribution vials.

Viability tests were then carried out using the same numbering technique on several freezers corresponding to each distribution bottle according to the schedule: D + 11 days, D + 1 month, D + 3 months, D + 6 months, D + 1 year . Identity

Identity is controlled from a freeze using ApiStaph® Gallery biochemical tests and a lysostaphin resistance test. These tests were carried out from an 18 hour culture at 37 ° C. on 2% NaCI Columbia agar and according to the indications given by the manufacturer.

On the other hand, 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.

An identity check of the capsular polysaccharides of the T5 and T8 strains was performed by slide agglutination using monoclonal antibodies specific for anti-PS5 (IgM) clone J1 and anti-PS8 (IgGI) clone K8-

24 obtained from hybridomas supplied by the Pasteur Institute which have been produced by conventional methods well known to those skilled in the art. The slide agglutination reactions are carried out from 50 .mu.l bacterial suspension adjusted to ₆ 8 ₀ DO = 5 nm.

Productivity assessment by ELISA Productivity test

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. At the end of culture, 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.

Stability test

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. For this, 10 ml of M1 are inoculated with an ampoule of a B-freeze and then, after 16 hours of culture at 37 ° C., 8 successive cultures are made by seeding 10 ml of medium M1 at OD = 0.2 and cultivating respectively during 4h, 4h, 16h, 4h, 4h, 16h, 4h and 4h at 37 ⁰ C. From the last culture is seeded 150 .mu.l on an MO medium inducing agar to perform a preculture at 37 ⁰ C for 16h, then inoculate 400 ml of M1 at OD = 0.2 and cultured for 72 hours at 37 ^° C. After the last pass on medium M1, a productivity measurement is carried out as above and compared to the productivity obtained for a reference frozen stock under the same conditions of culture.

Autoclave extraction and sample preparation

At the end of the culture, 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 μg / 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. PS5 ELISA Assay

Proteinase K treated samples (autoclave extracts and / or treated culture supernatants) 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. In the ELISA, 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 μg / ml. In the final step, 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.

Results

Congelates A CYL1892

After 11 days of freezing, the CYL1892 count results are stable. Four successive passages have been made. A total of 16 separate colonies were extracted and analyzed by PCR. All colonies tested at each passage have the type 5 cap locus and the presence of the capl promoter has been demonstrated for all colonies as well.

Congelats B CYU 892 After 11 days of freezing, the CYL1892 count results are stable (2.3x10 ⁹ CFU / ml on average).

Api Gallery

Proper identification of S. aureus is obtained by biochemical analysis as indicated by the manufacturer. PCR Profile The genetic stability of the pre-congelate was monitored during the various cloning steps. The presence of a specific amplification of capδ 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 capδ and cap1 promoter loci is observed.

Twenty isolated colonies from Frozen B CYL1892 were tested. The amplifications at about 500 bp and 200 bp illustrate the genetic stability of the capδ and cap1 promoter loci. Agglutination test

Only agglutination with the anti-PS5 monoclonal antibody is observed.

Productivity of Frozen A and B CYL1892 (Figure 1)

Productivity measurements were performed several times in parallel with references of frozen and frozen wild-type. As for the recombinant strains, two types of frozen were prepared for the wild strains. A Reynolds A freeze was obtained from the original strain by a small number of laboratory passages in 2% NaCl broth columbia. This Frozen A contains materials of animal origin. A second freeze (Reynolds B freeze), devoid of material of animal origin was obtained by successive passages of the original strain on media MO. The Reynolds B freezes were prepared in liquid MO medium after having undergone passages in non-inductive MO medium.

It is observed that 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 μg / ml and 30 μg / 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.

Stability of Frozen A and B CYL1892 (Figure 1) Productivity measurements were performed after a stability test on M1 medium (approximately 40 generations made). The productivity observed in the pellets and supernatants after several passages in M1 medium is equivalent to that obtained without passage for the frozen B CYL1892 as for the frozen A. A decrease in productivity of 20 to 50% is observed for the B-freezes of the strain Wild Reynolds.

Characterization of the S. aureus recombinant strain CYL1892 has shown that a productivity gain in capsular polysaccharides is obtained with respect to the use of the Reynolds wild-type strain. Surprisingly, the difference in productivity was essentially observed in the culture supernatant (approximately 50x, ie 200 μg / ml on average) and remained stable after successive passages in an inducing medium. These results show that the construction used for producing this recombinant strain is stable. Frozen A and B were therefore made from homogeneous populations.

Example 2 Characterization of the Type 5 Polysaccharide Capils Produced by the CYL1892 Strain and the Type 8 Capsular Polysaccharide Produced by the CYL770 Strain

The T8 and T5 rPS isolated and purified from the CYL770 and CYL1892 strains respectively were characterized by the analyzes described below.

One-dimensional proton magnetic resonance analysis of purified rPS5 and rPS8 showed that these polysaccharides have the same primary structure as previously published (C Jones, Carbohydr Res 2005, 340, 1097-1106). This analysis also makes it possible to show directly that rPS5 has a level of N- and O-acetylation greater than 70%. For the type 8 polysaccharide, the same analysis, carried out after de-O-acetylation of the antigen using NaOD added directly to the NMR tube, showed that the degree of N- and O-acetylation is greater than 70%. For 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).

Example 3 Preparation of an anti-CYL770 polyclonal serum adsorbed on chemically-acapsulated seeds and evaluation of its cross-reactivity on different S. aureus type 8 strains

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. on whole tryspsinated organisms in order to overcome the non-specific binding of antibodies via protein A, on the germs resulting from cultures of the recombinant strain CYL770 in medium M1 inductor. Anti-CYL770 serum was then tested under the same conditions on trypsinized whole cells of type 8 wild-type strains (Becker and Wright) derived from solid and liquid cultures of different levels of capsular polysaccharide induction.

Preparation of germs CYL770 for immunization A preculture is carried out in Erlenmeyer flask by seeding one ampoule (1 ml) of CYL770 freeze A in 9 ml of liquid MO medium. The pre-cultures are placed with x100 stirring at 37 ° C. for 18 h to 20 h. The strain CYL770 is then cultured in Erlenmeyer flask in 100 ml M1 by seeding at OD ₆₈₀ nm = 0.2 from the pre-culture made in liquid MO. The Erlenmeyer flask is then stirred x100 at 37 ° C. for 48 hours. The culture is then centrifuged, the pellet is taken up in 20 ml of PBS (stock solution) and the ODβsonm is measured. A volume of stock solution is taken to perform the count before formulation. From the collection of stock solution CYL770, dilutions of reason 10 are made in 24-well plate of 10 ^'1 to 10 ^{' 9} . Six drops of 25 μl of each dilution are seeded on Columbia 2% NaCl agar and incubated for 18 to 20 h at 37 ° C. CFUs are counted at dilutions where possible and the number of CFU / mL (X) is determined according to the formula: X = (average CFU per drop x 40) / read dilution. The determination of the equivalence DO / CFU (Y) is carried out as follows: for OD ₆ 80nm = 1, Y = (X / ODβδonm stock solution).

The formalin fixation is carried out for 15 hours by diluting a volume of CYL770 stock solution in PBS-3% formalin so as to obtain 80 ml of suspension at Dθ680nm = 1.2, according to the protocol described by (Karakawa et al., 1985 J Clin Microbiol 22: 445-447). After 15 hours of treatment, 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. 2% NaCl by spreading 150 μl of the amplified culture in a layer and incubation for 18 to 20 hours at 37 ^° C. The formalin-treated seeds then undergo three successive washes with 1X concentrated phosphate buffer (PBS) pH 7 in order to eliminate the formalin centrifugation for 30 min at 3500 rpm + ^{40 °} C. (2x 40 mL) ₁ wash 2x 40 mL, centrifuge 30 min at 3500 rpm + 4 ° C, washing 2x 30ml, centrifugation 30min at 3500rpm + 4 ° C, washing 2x 20ml (pool), centrifugation 30min at 3500rpm + 4 ° C, and recovery of germs in PBS. The suspension of germs thus treated can be stored at + ^40.degree .

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:

2x 4ml to 6x10 ⁷ CFU / ml (J ₂ + U injections)

4x 4ml to 1, 2x10 ⁸ CFU / ml (injections J ₀ + J ₇ + jg + Ji 1)

3x 4ml to 1, 8x10 ⁸ CFU / ml (injections ju + jiβ + jiβ)

3x 4ml at 2,4x10 ⁸ CFU / ml (injections J21 + J23 + J25) The caps are placed sterilely and the vials are crimped with forceps. Suspensions are stored at +4 ⁰ C a week.

Preparation of Chemically Acapsulated Germs for Adsorption

From an ampoule of the frozen A (1ml) of the strain CYL770, a preculture of 50ml of Columbia medium is seeded and incubated at 37 ° C with stirring for 18h. From the preculture, 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 pellet is then taken up in 8 ml of PBS and a measurement of OD is carried out: OD ₆₈₀ nm = 178.

The chemical treatment to be applied to the sprouts to remove capsular polysaccharides has been described by Karakawa et al. (1985 Karakawa et al., 1985 J Clin Microbiol 22: 445-447). The following treatment is carried out in duplicate: 4 ml of bacterial suspension at OD = 178 are transferred into a 250 ml glass Schott flask, 30 ml of 0.2M HCl glycine 0.14M pH 2 are added and the mixture is incubated for 20 min at 100 ⁰ C (water bath). The suspension is transferred to 50ml Falcon tubes, centrifuged at 3500 rpm for 30 min at + ^{40 °} C. and the supernatant removed. The pellets are washed 3 times with 40ml of PBS. 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. After the fifth cycle, the adsorbed serum is recovered and filtered through a 0.22 μm 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. To prepare the trypsinized seeds, cultures in solid MO medium (inducing agar (for type 8 strains) and Columbia 2% NaCl (for the Reynolds strain) were carried out as follows: 900 μl of PBS are added to 100 μl of frozen Becker, Reynolds and Wright and 150μl of bacterial suspension are plated in stem web on a petri dish and incubated at 37 ⁰ C for 18h (37 ⁰ C + 5% CO ₂ for the Becker strain). The tablecloths are then taken up in PBS and the suspensions are adjusted to

Dθ680nm ⁼ 5. For the Becker and Wright type 8 strains, 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.

The bacterial suspensions resulting from the solid and liquid cultures and adjusted to DOeβ are treated with trypsin so as to eliminate any troublesome trace of protein A. For this, from an aliquot of bacterial suspension of 5ml, 100μl 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 pellets were washed 3 times with 5 ml of PBS and taken up again with the necessary volume of PBS in order to to adjust the suspensions to DOβsonm = 5. The suspensions are stored at +4 ^° C. until use.

The agglutination tests were carried out with 50 μl of fresh seeds trypsinated at OD _680n m = 5. The seeds were spread on the slide so as to completely cover the bounded glass circle intended for agglutination without outflowing outwards. . 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 μl of anti-CYL770 polyclonal serum adsorbed on chemically decapsulated seeds was used for the agglutination tests. After depositing the serum on the slide in the three wells, 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.

Results

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).

Table 2: Concentration of Formulas CYL770 Germs for Immunization

Once produced, the serum is adsorbed on chemically-activated autologous seeds and then evaluated against type 8 strains in slide agglutination (Table 3).

Table 3: Evaluation of adsorbed anti-CYL770 serum in slide agglutination

Becker CYL770 Wright Reynolds

MO M1 TSB M1 MO M1 TSB Agar Agar Agar Agar Inducer Inducer

PBS - - - - - - - -

Serum +++ +++ +++ ++ +++ ++ _ anti

CYL770 adsorbed

Mab-PS8 ++ (+) +++ - ++++ ++++ ++++ ++ -

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

+ total agglutination observed in 3 to 5 min

+/- Very slow and total agglutination observed in 10 min no observed agglutination

Agglutination of Becker strains (except in TSB medium), Wright and CYL770 with adsorbed anti-CYL770 serum was observed. Furthermore, this serum does not allow the agglutination of Reynolds control germs (type 5) cultured in reference medium Columbia 2% NaCl agar, or Becker germs grown in TSB medium inducing PS8 thus demonstrating its specificity.

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.

It thus appears that the adsorbed anti-CYL770 serum thus produced allows cross-reactivity on wild-type germs having a type 8 capsule. Example 4 Generation of polyclonal anti-PS8 antibodies in the rabbit from strain CYL770

To characterize the nature of the PS8 produced by the strain CYL770, 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

(Karakawa et al., 1985, J. Clin Microbiol., 22: 445-447).

Injections: Three female rabbits New Zealand White (ESD - Charles River

Laboratories, St Germain-sur-1'Arbresle, France) of 2.5 kg are injected 3 times a week for 4 weeks with different concentrations of inactivated whole germs of strain CYL770 grown in M1 medium and dialysed against PBS. The first injection (OJ) is performed subcutaneously in the scapular girdle to avoid septic shock. All subsequent injections are made intravenously into the marginal vein of the ear. The concentrations and volumes used for each injection are described below.

Table 4: Group Description

Groups Antigen Buffer Route Volume Dilution Injection

AT . poo 1 v SC for the first injection

1 rabbit ^PBS H 7 2 ^{IV for all others 1 ml}

^ P 'injections

CYL770 in PBS 1X, SC for the first injection

2: medium M1 pH 7.2 IV for all other ₁ , 2 rabbits 0.6 to 2.4x10 ⁸ injections CFU / rabbit Composition of PBS 1X pH 7.2: 136 mM NaCl; 2.7 mM KCl; 8 mM

Na ₂ HPO ₄ ; 7.5 mM KH ₂ PO ₄

Blood collection from preimmune animals: Five to 10 ml of blood are collected in Vacutainer ™ 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 ™ 50ml tubes.

All sera are stored at -20 ^° C. until use

Table 5: Immunization Diagram

Analyzes of anti-PS8 antibody responses

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) are incubated overnight at room temperature (20-22X) in the presence of 1 μg purified PS8 per ml in 1X PBS pH 7.2 ( 100 μl / well). The plates are washed 4 300 μl / 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 μl / well of PBS-Tween / 1 % BSA. The plates are then washed 4 times according to the method described above. 100 μl 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)

Inc. Birmingham) is diluted 1/12000 in PBS-Tween / 1% BSA and 100 μl of the conjugate solution is added per well. The plates are incubated for 1 h 30 at 37 ° C. and then washed 4 times with PBS-Tween. For the revelation, 100 μl of the ready-to-use TMB substrate solution (TEBU Bio-laboratories) are added per well and the plates are incubated for 15 min at room temperature (20-22 ^° C.) and in the dark. . The enzymatic reaction due to peroxidase is stopped by the addition of 100 μl of H ₃ PO ₄ 1 M per well. The optical density (OD) of each of the wells is measured at 450 nm using an automatic plate reader (Versamax). The background noise (average value over 4 white wells) is subtracted from the measured OD values.

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.

Table 6: Anti-PS8 IgG Titres

serums

Becker M1 CYL770 M1 CYL770 M1 Positive Control

Anti-PS8 IgG Titers 30,000 8,500 5,000 Example 5: Production of Conjugates

5.1- Production of type 8 conjugates (STAPH8-rEPA)

Depolymerization of purified polysaccharide type 8 from strain CYL770 Before conjugation, 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 μl of 200 mM ascorbic acid, 11 μl of 20 mM CuSO4 and 11 μl 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.

Activation of the antigen

40 mg of antigen (depolymerized for polysaccharide purified from strain CYL770) was dissolved in 8 ml of 200 mM NaCl and 125 mM adipic acid dihydrazide (ADH). The pH is adjusted to 4.9 and ethyldimethylaminopropylcarbodimide (EDAC) is added to a final concentration of 25 mM. During the course of the activation which lasted 90 min at room temperature, 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 Δ553 ie rEPA) in 100 mM NaCl and 50 mM EDAC was prepared. During the course of the conjugation which lasted 90 min at 4 ° C, the pH is constantly adjusted to a value of 5.6 by adding a dilute solution of HCl. At the end of the reaction, 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.

Characterization of 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 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.

5.2- Production of conjugates of type 5 (STAPH5-rEPA)

Depolymerization of purified polysaccharide type 5 from strain CYL1892

Prior to conjugation, the rPS5 antigen purified from strain CYL1892 is depolymerized according to the principle described in US Pat. No. 6,045,805.

The rPS5 antigen is prepared at 2 mg / ml. To 20 ml of this solution at room temperature are added 55 μl of 200 mM ascorbic acid, 5.5 μl of 20 mM CuSO4 and 5.5 μl 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

40 mg of antigen (depolymerized for polysaccharide purified from strain CYL1892) were dissolved in 8 ml of 200 mM NaCl and 50 mM adipic acid dihydrazide (ADH). The pH is adjusted to 4.9 and ethyldimethylaminopropylcarbodimide (EDAC) is added to a final concentration of 10 mM. During the course of the activation which lasted 45 min at room temperature, 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. During the course of the conjugation which lasted 90 min at 4 ° C, the pH is constantly adjusted to a value of 5.6 by adding a dilute solution of HCl. At the end of the reaction, 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.

Characterization of 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

HPSEC / LS / RIA / isc. Example 6 Evaluation of the Immunogenicity of Conjugates of Type 8

(PS T8-rEPA) in the mouse

Different batches of PS T8-rEPA conjugates using PS8 purified from Becker type 8 strain were generated by the authors.

The immunogenicity of these 8-type conjugates has been demonstrated in different mouse lines, inbred or non-consanguineous, by the analysis.

ELISA of the anti-PS8 humoral response (IgM, IgG and IgG subclasses). Dose-effect studies have also been performed on this type of animal model.

To demonstrate the immunogenicity induced by rPSfT8-rEPA conjugates generated using PS8 purified from recombinant strain CYL770, animal tests and immunological assays of the same type as those developed for type 8 conjugates. having a Becker origin (wild strain), have been developed and implemented.

Immunization protocol

Injections: 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.

Intermediate blood sampling: Approximately 500 μl of blood are collected at the retro-orbital sinus and collected in Vacutainer ™ tubes in pre-immune animals, before the "prime" (OJ) and then at D21, before amplification ("). boost ") for each mouse. For the intermediate samples, the 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.

Final blood collection by exsanguination: All animals are exsanguinated under general anesthesia at day 35, 15 days after amplification. Between 1 ml and 1.5 ml of blood are collected per animal and collected in Vacutainer ™ tubes.

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 μg / ml.

PS8 purified from Becker strain, and not purified from recombinant strain CYL770, was chosen for adsorption to evaluate the antibody response generated by rPSfT8-rEPA conjugates (origin CYL770) against sPS8 purified from a so-called wild strain. All sera specific for PS8 are analyzed by robotic ELISA (Zymark robot) according to the following procedure.

96-well ELISA plates (M129B Dynex) are incubated overnight at room temperature (20-22 ^° C.) in the presence of 1 μg / ml of purified PS8 starting from the Becker strain in 1X PBS pH 7.2 (100 μl). / well). The plates are washed 4 times with 300 μl / 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 μl / well of PBS-Tween / 1% BSA. The plates are washed 4 times according to the method described above. 100-μl per well of each of the sera to be tested at different concentrations are deposited in the plate. Successive dilutions of reason 2 are carried out within the plate (in-line dilution) in PBS-Tween 1% BSA. The plates are incubated for 1 hour at 37 ° C. and then washed 4 times with PBS-Tween 100 μl / well of anti-mouse Ig conjugate, diluted to the appropriate concentration according to the class of Ig in PBS-Tween. 1% BSA are added and the plates are incubated for 1 h at 37 ^° C. and then washed 4 times with PBS-Tween. For the revelation, 100 μl of the TMB solution is added per well and the plates are incubated for 20 min at room temperature (20-22 ^° C.) and in the dark. 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-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 Ig titre of this standard, expressed in arbitrary ELISA unit, was previously determined on a plurality of several measurements and corresponds to the arithmetic average of the dilution inverses for an OD45 ₀ nm = 1 - the detection threshold of this ELISA is evaluated to 10 ELISA units (1log-ι ₀ ). The titres of each serum are expressed in log-ι ₀ .

Results

Title of anti-PS8 IgM

The results shown in FIG. 2 show that a significant but non-specific anti-PS8 response is observed in all preimmune animals (titre greater than 2 log). After sensitization (J21), a marked increase in the anti-PS8 IgM specific response was observed for the 4 conjugate groups (~ + 2 Log) and no difference was observed between these 4 groups. For groups receiving unconjugated PS8, there was a marked increase in IgM anti-PS8 titres for group A (Becker's PS8 + unconjugated rEPA) of about 1 Log and for group G (native PS8 of CYL770 + rEPA, unconjugated) of about 1.5 log. No increase in anti-PS8 IgM titers was observed for group B (fractionated PS8 of CYL770 + unconjugated rEPA). At J35, there is no significant amplification effect of the IgM response regardless of the group considered. Total anti-PS8 IgG titre

The results shown in Figure 3 show that no or very little non-specific anti-PS8 IgG response is observed in preimmune animals (<1.3 Log). After sensitization (J21), a very strong anti-PS8 IgG specific response was observed for the 4 conjugate groups (> + 2.3 Log), with response averages between 3.5 Log and 4.1 Log. No differences were observed between the 4 conjugate groups after sensitization. This anti-PS8 response is significantly greater than that obtained in the groups injected with the non-conjugated PS. However, there is an increase of the anti-PS8 IgG titers at D21 of about 1 log for groups A (Becker's PS8 + unconjugated rEPA) and G (native PS8 of CYL770 + unconjugated rEPA). No increase in anti-PS8 IgG titers was observed for group B (fractionated PS8 of CYL770 + unconjugated rEPA).

At J35, a significant enhancer effect is observed only for the 4 groups of conjugates regardless of the group considered (> + O.ΘLog). The averages of anti-PS8 responses are between 4.1 Log and 4.9Log. Considering the response averages, there is no significant difference between the Becker conjugates and the CYL770 conjugates. However, it seems that the anti-PS8 response obtained with the conjugates from strain CYL770 evaluated on PS8 purified from the Becker wild-type strain, is more homogeneous within the same group.

Titers of IgGI subclasses, IgG2a and Ig3 anti-PS8, measured on day 35 Figure 4 shows that no non-specific anti-PS8 response is observed in preimmune animals (<1.2 Log).

For IgGI, a very strong anti-PS8 specific response is observed for the 4 conjugate groups (> 4 Log) while no response is detected for the 3 groups that received the different types of non-conjugated polysaccharides. There is no significant difference in the mean of the responses between Becker conjugates and CYL770 conjugates, but it seems nevertheless that the response obtained with the 2 CYL770 conjugates is more homogeneous within the same group.

For IgG2a, 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. For 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

These results show that the PS8-rEPA conjugates, generated from purified PS8, derived from the so-called wild-type Becker strain or from the recombinant CYL770 strain, induce strong anti-PS8 specific antibody responses in the OF1 mouse model, compared to the purified polysaccharides. injected alone. A significant enhancer effect is also observed for these four batches of conjugates. This response is evaluated on PS8 purified from the so-called wild Becker strain. From the point of view of the averages of the antibody responses, no significant difference is observed between the different batches of conjugates, whatever the origin of the PS8. However, it appears that the total IgG and IgGI anti-PS8 responses obtained with the CYL770 conjugates are more homogeneous within the same group. Example 7 Evaluation of the Immunogenicity of Conjugates of Type 5 (PS T5-rEPA in Mice

Different batches of PS T5-rEPA conjugates were generated using purified PS5 from the Reynolds strain or purified rPS5 from the Staphylococcus aureus recombinant strain CYL1892.

The immunogenicity of these type 5 conjugates could be demonstrated in the mouse, by an ELISA analysis of the anti-PS5 antibody response (total IgM and IgG). To compare the specific antibody responses induced, each conjugate was injected at an optimal dose of immunization (previously defined in dose-effect studies) of 2.5 μg / mouse / injection).

Immunization protocol

Injections: 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 μg / 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 8: Characteristics of conjugates tested

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

Intermediate blood samples: Approximately 200μl of blood is collected from the retroorbital sinus and collected in Vacutainer ™ tubes. These samples are taken at OJ on the preimmune animals before sensitization, D21 before boosting ("boost") for each mouse individually. For the intermediate samples, the 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).

Final blood collection by exsanguination: All animals are exsanguinated by carotid section under general anesthesia at day 35, 15 days after the day of amplification. Between 1ml and 1.5ml of blood are collected per animal and collected in Vacutainer ™ tubes.

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.

Analysis of the antibody response

The 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 μg / 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. wild Staphylococcus aureus. 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 μg / ml of purified sPS5. from the Reynolds strain in 1X PBS pH 7.2 (100 μl / well). The plates are washed 4 times with 300 μl / 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 μl. / well of PBS-Tween / 1% BSA. The plates are washed 4 times according to the method described above. 100-μl per well of each of the sera are deposited in the plate. Serial 2-fold dilutions of the sera are performed within the plate (in-line dilution) in PBS-Tween 1% BSA. The plates are incubated for 1 hour at 37 ° C. and then washed 4 times with PBS-Tween 100 μl / well of anti-mouse Ig conjugate, diluted to the appropriate concentration according to the class of Ig in PBS-Tween. 1% BSA, are added and the plates are incubated for 1 h at 37 ° C. and then washed 4 times with PBS-Tween. For the revelation, 100 μl of the TMB solution are added per well and the plates are incubated for 20 min at room temperature (20-22 ^° C.) and in the dark. 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. The Ig titre of this standard, expressed in arbitrary ELISA unit, was previously determined on a plurality of several measurements and corresponds to the arithmetic average of the dilution inverses for an OD ₄₅₀ nm = 1. The detection threshold of this ELISA is evaluated. to 10 ELISA units (1log-ιo). The titres of each serum are expressed in log-io.

ResultsTitles of anti-PS5 IgM

The results shown in Figure 7 (a) show that a significant but non-specific anti-PS5 response is observed in all preimmune animals (mean value of the titles ~ 1.8 Log ± 0.2 Log). Mice injected with PS5 or purified non-rEPA-conjugated rPS5 showed a lower anti-PS5 IgM response at D21 (-0.7 Log) and at D35 (-1.2 Log) than mice with injected with conjugates.

A specific and significant response was observed for the 4 conjugates at D21 and D35, with a mean value of titers obtained around 3.2 log ± 0.2 log and 4 log ± 0.2 log, respectively.

No significant difference was found between the different conjugates in terms of anti-PS5 IgM response at D21 and D35, they therefore have immunogenicity similar to that of PS5-rEPA conjugates. In addition, 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.

Titers of anti-PS5 IgG

The results presented in Figure 7 (b) show that no anti-PS5 IgG response is observed in preimmune animals (mean value of titres <1, 3 Log: detection threshold).

A significant anti-PS5 IgG response was observed on D21, for the groups of mice injected with either conjugate, with a mean value of the titers evaluated around 3.2 Log ± 0.2 Log . A very strong "boost" effect is observed at day 35 for all the groups of mice injected with one or other of the conjugates, with a mean value of the titers evaluated around 5 log ± 0.2 log.

In addition, no significant difference was found at J21 as at J35 between the 4 conjugates so the rPS5-rEPA conjugates show immunogenicity similar to that of the PS5-rEPA conjugates. In addition, 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. In addition, these studies have also demonstrated that the 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.

Claims

claims

1. Immunogenic composition comprising a type 8 capsular polysaccharide and or type 5 of a T8 and / or T5 supramuscular S. aureus strain respectively.

2. An immunogenic composition according to claim 1 comprising a type 5 capsular polysaccharide of a strain of S. aureus CYL1892.

An immunogenic composition according to claim 1 or 2 comprising a type 8 capsular polysaccharide of a strain of S. aureus CYL770.

An immunogenic composition comprising a conjugate comprising a T8 and / or T5 capsular polysaccharide of an overproducing strain of S. aureus bound to a carrier protein.

An immunogenic composition according to claim 4, wherein the conjugate comprises a capsular polysaccharide T8 of strain CYL770.

The immunogenic composition of claim 4, wherein the conjugate comprises a T5 capsular polysaccharide of strain CYL1892.

An immunogenic composition according to claim 4 comprising a conjugate comprising a capsular polysaccharide T8 of strain CYL770 and a conjugate comprising a T5 capsular polysaccharide of strain CYL1892.

8. Immunogenic composition according to any one of claims 4 to 7, wherein the carrier molecule is exotoxin A Pseudomonas aeruginosa.

The immunogenic composition of any one of claims 4 to 8, wherein the T5 and / or T8 capsular polysaccharide is fractionated.

10. Conjugate comprising a T5 or T8 capsular polysaccharide of an overproducing strain of S. aureus bound to a carrier protein.

11. Conjugate according to claim 10 wherein the capsular polysaccharide is a T5 PS of the strain of S. aureus CYL1892.

12. Conjugate according to claim 10 wherein the polysaccharide is a T8 PS of the S. aureus CYL770 strain.

13. Conjugate according to any one of claims 10 to 12, wherein the carrier molecule is exotoxin A Pseudomonas aeruginosa.

14. Conjugate according to any one of claims 10 to 12 wherein the PS is fractionated.

15. T5 capsular polysaccharide isolated or purified from S. aureus strain CYU 892.

16. Use of a conjugate according to any of claims 10 to 14 for the manufacture of an immunogenic composition for immunization against S. aureus infection.

17. A process for producing T5 capsular polysaccharide comprising the steps of: a) culturing an overproducing strain of S. aureus, b) inactivating the culture thus produced, and c) recovering the T5 capsular polysaccharide from the supernatant.

The method of claim 17 wherein the overproducing strain is CYL1892.