PROCESSING PROCESS

专利摘要:
La demande décrit en outre une composition immunogène comprenant ; (i) un saccharide capsulaire de Type 5 de S. aureus conjugué à une protéine porteuse, (ii) un saccharide capsulaire de Type 8 de S. aureus conjugué à une protéine porteuse, (iii) une protéine ClfA ou un fragment immunogène de celle-ci, (iv) une anatoxine alpha, et (v) un excipient pharmaceutiquement acceptable ; dans laquelle le pH de la composition immunogène est pH 5,0 à pH 8,0. The application describes an immunization method against Staphylococcus aureus infections comprising a step of administering to a human patient a single dose of an immunogenic composition comprising; (i) a Staphylococcus aureus ClfA protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg and (ii) a pharmaceutically acceptable excipient; wherein the pH of the composition is pH 5.0 to pH 8.0. The application further discloses an immunogenic composition comprising; (i) a S. aureus Type 5 capsular saccharide conjugated to a carrier protein, (ii) a S. aureus Type 8 capsular saccharide conjugated to a carrier protein, (iii) a ClfA protein or an immunogenic fragment of that (iv) an alpha toxoid, and (v) a pharmaceutically acceptable excipient; wherein the pH of the immunogenic composition is pH 5.0 at pH 8.0.
公开号:BE1023004B1
申请号:E2015/5796
申请日:2015-12-08
公开日:2016-10-31
发明作者:Ralph Leon Biemans；Dominique Boutriau；Philippe Denoel；Pierre Duvivier；Carine Goraj
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

PROCESSING METHOD
Technical area
The present invention relates to the field of immunogenic compositions and staphylococcal vaccines, their manufacture and the use of these compositions in medicine. More particularly, it relates to the use of a ClfA protein (Agglutinating Factor A) or a fragment thereof (preferably an immunogenic fragment thereof) from S. aureus. These ClfA proteins or fragment thereof can be combined with other staphylococcal proteins and / or capsular saccharides derived from S. aureus antigens to form multivalent compositions.
Context
Staphylococcus aureus (S. aureus) are commensal Gram-positive bacteria that colonize the nostrils, armpits, pharynx and other mucosal and cutaneous surfaces of up to 30% of human subjects. It is estimated that S. aureus is responsible for 20 to 25% of all care-associated infections (Wisplinghoff et al Clin Infect Dis, 2004; 39; 309-317), tripling the length of hospital stays and by 5 the risk of death in hospital for infected patients compared to patients not affected by these infections (Noskin et al., Intern, Med 2005; 165; 1756-1761). S. aureus infections can be associated with in-hospital mortality rates of up to 25%. Historically, S. aureus has been associated primarily with nosocomial infections. The severity of these infections has increased due to the recent dramatic increase in S. aureus infections associated with antibiotic resistance. Staphylococcus aureus is the most common cause of nosocomial infections with high morbidity and mortality (Romero-Vivas et al 1995, Infect Dis 21, 1417). It is the cause of some cases of osteomyelitis, endocarditis, septic arthritis, pneumonia, abscess and toxic shock syndrome. Passive immunotherapy involving the administration of polyclonal antisera directed against staphylococcal antigens was investigated (WO 00/15238, WO 00/12132), as well as immunotherapy using a monoclonal antibody directed against the acid lipoteichoic (WO 98/57994). However, to date, none have been licensed for use. Several candidate immunotherapies have failed to prove their efficacy in humans. These include: Altastaph (Nabi Biopharmaceuticals), which contains purified CP5 and CP8 antibodies from subjects vaccinated with StaphVAX ™ (experimental vaccine developed and licensed by Nabi Biopharmaceuticals, Rockville, MD, USA, Veronate (Inhibitex), polyclonal antibodies targeting S. aureus agglutination factor (ClfA) and S. epidermidis adhesion SdrG protein; Aurexis (Tefibazumab, Inhibitex), monoclonal antibodies targeting ClfA; Aurograb (NeuTec Pharma), single chain antibodies against a β-linkage cassette transporter and Pagibaximab (Biosynexus), a monoclonal anti-lipoteichoic acid antibody (Dejonge et al J.Pediatrics 2007; 151; 260-265; Rupp et al Antimicrob. Agents Chemother. 2007; 51; 4249 -4254).
Active vaccination to generate a polyclonal immune response against staphylococci has also been investigated. One approach reported in WO 03/61558 uses Type 5 and Type 8 capsular polysaccharide conjugates of S. aureus conjugated to Pseudomonas exoprotein A (StaphVAX - Nabi Biopharmaceuticals). Another approach used an S. aureus IsdB protein (V710-Merck & Co) but failed to demonstrate efficacy (Fowler et al 2013, JAMA 309, 1368-1378).
Description of figures
Figure 1 - Percentage of subjects complaining of pain after 1 or 2 doses of the 4-component vaccine (4C). In each grouping of formulation, the first three columns indicate the% of subjects complaining of pain after administration of a single dose, the first column representing all reports of pain, the second column representing a pain greater than or equal to level 2 and the third column represents a level 3 pain. The 4th, 5th and 6th columns indicate the same information after administration of the second dose.
Figure 2 - Percentage of subjects with redness after 1 or 2 doses of 4C vaccine. In each grouping of formulation, the first three columns indicate the% of subjects presenting redness after administration of a single dose, the first column representing all the reports of redness, the second column representing more than 50mm of redness and the third column representing more than 100mm of redness. The 4th, 6th, and 6th gages indicate the same information after administration of the second dose.
Figure 3 - Percentage of subjects with swelling after 1 or 2 doses of the 4C vaccine. In each formulation group, the first three columns indicate the% of subjects with swelling after a single dose, the first column representing all reports of swelling, the second column representing more than 50mm swelling and the third column representing more than 100mm of swelling. The 4th, 5th and 6th columns show the same information after the second dose.
Figure 4 - Immunogenicity results for antibodies against S. aureus type 5 capsular polysaccharide. The results of the Geometric Mean Antibody Concentration (GMC) of a Luminex assay detecting antibodies against Type 5 capsular polysaccharide at various time points after the first and second immunizations are illustrated. The chosen time points are day 0 before immunization, day 7 after immunization, day 14 after immunization, day 30 after immunization, day 7 after two immunizations (corresponding to day 37 on the graph), day 14 after two immunizations (corresponding to day 44 on the graph) and day 30 after two immunizations (corresponding to day 60 on the graph). For each point in time, the results are presented in order (from left to right) of, 5/10, 5 / 10AS, 10/30, 10 / 30AS and saline.
Figure 5 - Immunogenicity results for antibodies to Type 8 capsular polysaccharide of S. aureus. The GMC results of a Luminex assay detecting antibodies to type 8 capsular polysaccharide at various time points after the first and second immunizations are illustrated. The chosen time points are day 0 before immunization, day 7 after immunization, day 14 after immunization, day 30 after immunization, day 7 after two immunizations (corresponding to day 37 on the graph), day 14 after two immunizations (corresponding to day 44 on the graph) and day 30 after two immunizations (corresponding to day 60 on the graph). For each point in time, the results are presented in order (from left to right) of 5/10, 5/1 OAS, 10/30, 10 / 30AS and saline.
Figure 6 - Immunogenicity results for antibodies to S. aureus alpha toxoid. The GMC results of a Luminex assay detecting antibodies against alpha toxoid at various points in time after the first and second immunizations are illustrated. The chosen time points are day 0 before immunization, day 7 after immunization, day 14 after immunization, day 30 after immunization, day 7 after two immunizations (corresponding to day 37 on the graph), day 14 after two immunizations (corresponding to day 44 on the graph) and day 30 after two immunizations (corresponding to day 60 on the graph). For each point in time, the results are presented in order (from left to right) of 5/10, 5/1 OAS, 10/30, 10 / 30AS and saline.
Figure 7 - Immunogenicity results for S. aureus ClfA antibodies. The GMC results of an ELISA test detecting antibodies against ClfA at various points in time after the first and second immunizations are illustrated. The chosen time points are day 0 before immunization, day 7 after immunization, day 14 after immunization, day 30 after immunization, day 7 after two immunizations (corresponding to day 37 on the graph), day 14 after immunizations (corresponding to day 44 on the graph) and day 30 after two immunizations (corresponding to day 60 on the graph). For each point in time, the results are presented in order (from left to right) of 5/10, 5 / 10AS, 10/30, 10 / 30AS and saline.
Figure 8 - Immunogenicity results for S. aureus type 5 capsular polysaccharide (panel A), S. aureus type 8 capsular saccharide (panel B), alpha toxoid (panel C) and ClfA (panel D) over a longer period of time from day 0 to day 540, after 1, 2 or 3 immunizations.
detailed description
Many problems are associated with the development of a vaccine against S. aureus infections. The failure of single-component vaccines (capsular polysaccharide or IsdB protein) suggests that a more complex vaccine containing multiple components may be needed to induce protective immunity. However, the combination of different antigens in an immunogenic composition may give rise to interferences in the composition (Skurnik et al (2010) J Clin Invest 120, 3220-3233). The identification of components to be combined in a multivalent composition is therefore not obvious. There is still a need to develop an effective vaccine against staphylococcal infections, particularly in view of the increased frequency of multidrug-resistant strains.
In the case of immunization against nosocomial staphylococcal infections, immunization can often take place shortly before hospitalization or surgery or placement of a permanent catheter. It would therefore be advantageous to achieve high levels of immunity with a single immunization. The use of lower doses of conjugate also has the advantages of a relative efficiency of vaccine production and associated economic benefits.
Accordingly, there is provided an immunization method against Staphylococcus aureus infections comprising a step of administering to a human patient a single dose of an immunogenic composition comprising; (i) a Staphylococcus aureus ClfA protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg and (ii) a pharmaceutically acceptable excipient; wherein the pH of the composition is pH 5.0 to pH 8.0.
In a second aspect of the present invention there is provided an immunogenic composition comprising; (i) a Staphylococcus aureus ClfA protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40, and (ii) a pharmaceutically acceptable excipia; wherein the pH of the immunogenic composition is pH 5.0 to 8.0, for use in the treatment or prevention of Staphylococcus aureus infections where a human patient is immunized with a single dose of the immunogenic composition.
In another aspect of the present invention there is provided an immunization method against Staphylococcus aureus infections comprising a step of administering to a human patient a single dose of an immunogenic composition comprising; (i) a Staphylococcus aureus alpha toxoid protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40, and (ii) a pharmaceutically acceptable excipient, wherein the immunogenic composition has a pH of pH 5.0 to pH 8.0.
In another aspect of the present invention there is provided an immunogenic composition comprising; (i) a Staphylococcus aureus alpha toxoid protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg, and (ii) a pharmaceutically acceptable excipient; wherein the pH of the immunogenic composition is pH 5.0 to pH 8.0, for use in the treatment or prevention of Staphylococcus aureus infections where a human patient is immunized with a single dose of the immunogenic composition.
In another aspect of the present invention there is provided an immunogenic composition comprising; (i) a S. aureus Type 5 capsular saccharide conjugated to a carrier protein, (ii) a S. aureus type 8 capsular saccharide conjugated to a carrier protein, (iii) a ClfA protein or an immunogenic fragment of that (iv) an alpha toxoid, and (v) a pharmaceutically acceptable excipient; wherein the pH of the immunogenic composition is pH 5.0 at pH 8.0.
In another aspect of the present invention there is provided a vaccine comprising a S. aureus type 5 capsular saccharide conjugated to a carrier protein, a S. aureus Type 8 capsular saccharide conjugated to a carrier protein, a ClfA protein or a an immunogenic fragment thereof and an alpha toxoid and a pharmaceutically acceptable excipient, wherein the pH of the immunogenic composition is pH 5.0 at pH 8.0.
In another aspect of the present invention there is provided a method for making the immunogenic composition or vaccine of the present invention comprising the steps of: a) conjugating a S. aureus Type 5 capsular saccharide to a carrier protein to form a conjugate S. aureus type 5 capsular saccharide, b) conjugate a S. aureus Type 8 capsular saccharide conjugated to a carrier protein to form a S. aureus type 8 capsular saccharide conjugate, and c) combine the conjugate. S. aureus type 5 capsular saccharide, S. aureus type 8 capsular saccharide conjugate, a ClfA protein or immunogenic fragment thereof and alpha toxoid to form the immunogenic composition; wherein the pH of the immunogenic composition is pH 5.0 at pH 8.0.
The present invention describes a method of immunization against Staphylococcus aureus infections comprising a step of administering to a human patient a single dose of an immunogenic composition comprising; (i) a Staphylococcus aureus ClfA protein or an immunogenic fragment thereof at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg and (ii) a pharmaceutically acceptable excipient; wherein the pH of the composition is pH 5.0 to pH 8.0.
Agglutination factor A (ClfA) has been identified as a S. aureus fibrinogen binding protein (US6008341) and has been identified as a potential carrier protein for polysaccharides that could be used to immunize against staphylococcal infections (WO 04 / 80490). ClfA is a surface-located protein and is an important virulence factor because of its fibrinogen binding and contribution to S. aureus adhesion. ClfA contains a fibrinogen binding region. This region, known as the A domain, is located towards the N-terminus of ClfA and comprises three separately folded subdomains known as N1, N2 and N3. Domain A is followed by a serine-aspartate repetition region and a region covering the wall and cell membrane that contains the LPXTG motif for cell wall anchoring promoted by the sortase. ClfA binds to the C-terminus of the γ chain of fibrinogen, and is thus able to induce agglutination of bacteria in a fibrinogen solution (McDevitt et al (1997) Eur J. Biochem 247; 416-424). Amino acid residues 221-559 of ClfA correspond to the N2-N3 region which retains binding of fibrinogen. The fragments containing amino acids 221-559 of ClfA are fragments which are suitable for use in the context of the invention. Amino acid residues 532 to 538 correspond to the ClfA locking peptide region. Each subdomain comprises nine β strands that form a new IgG fold. The peptide binding site of the chain of fibrinogen in ClfA is located in a hydrophobic groove at the junction between N2 and N3. Recently, amino acids P336 and Y338 of ClfA have been recognized as fibrinogen binding sites, the mutation of which has led to the loss of fibrinogen binding (Josefsson et al., 2008, PLOS One Volume 3, Issue 5, page 1-7 ). SEQ ID NOS: 8 to 12, 17 and 18 contain point mutations at positions 336 and 338. The loss of fibrinogen binding in these variants has led to a greater ability to protect against death following sepsis in immunized mice, which made it possible to conclude that the vaccine potential of recombinant ClfA is improved by eliminating its ability to bind fibrinogen (WO 09/95453). However, variants with single point mutations among Y256, P336, Y338 or K389 also lose their ability to bind fibrinogen (Deivanayagam et al EMBO J, 21; 6660-6672 (2002)). These unique point mutations should exhibit similarly enhanced immunogenicity, so simple mutations may also be used within the scope of the invention. In one embodiment, the immunogenic composition further comprises a ClfA protein or an immunogenic fragment thereof. The ClfA protein or immunogenic fragment thereof is optionally recombinant, isolated or purified.
In one embodiment, the ClfA protein is at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identical to the SEQ ID polypeptide sequence. N °: 3, 4, 5, 6 or 7 or 8 to 12 along the entire length thereof. "Identity" as known in the art, is a relation between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also refers to the degree of relatedness of the sequences between the polypeptide or polynucleotide sequences, as the case may be, as determined by the correspondence between the chains of these sequences. "Identity" can easily be calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects , Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humaina Press, NJ, 1994; Sequence Analysis in Molecular Biology , von Heine, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, and Carillo, H., and Lipman, D. , SIAM J. Applied Math., 48: 1073 (1988)). The methods for determining the identity are intended to give the greatest match between the sequences tested. In addition, methods for determining identity are codified in publicly available computer programs. Methods using computer programs to determine the identity between two sequences include, but are not limited to, the GAP program of GCG (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)). , BLASTP, BLASTN (Altschul, SF et al., J. Molec Biol., 215: 403-410 (1990)), and FASTA (Pearson and Lipman Proc Natl Acad Sci USA 85, 2444-2448 (1988)). )). The BLAST program family is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894, Altschul, S., et al., J. Mol .
Biol. 215: 403-410 (1990)). The famous Smith Waterman algorithm can also be used to determine identity.
The parameters applicable to the comparison of the polypeptide sequences include the following:
Algorithm: Needleman and Wunsch, Mol Mol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 by Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89: 10915 to 10919 (1992) Gap Penalty: 8 Gap Length Penalty: 2
A useful program with these parameters is publicly available under the name "gap program" from the Genetics Computer Group, Madison Wl. The above parameters are the default parameters applicable to peptide comparisons (as well as the absence of penalty for end gaps).
The parameters applicable to the comparison of polynucleotides include the following:
Algorithm: Needleman and Wunsch, Mol Mol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3
Available under the name "Gap Program" from the Genetics Computer Group, Madison Wl. These are the default settings for nucleic acid comparisons.
When a protein is specifically mentioned in the present description, it encompasses native or recombinant full length protein or optionally a mature protein from which any signal sequence has been removed. The protein can be isolated directly from the staphylococcal strain or produced by recombinant DNA techniques. Immunogenic fragments of the protein may be incorporated into the immunogenic composition according to the invention. These are fragments comprising at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids or at least 100 amino acids, taken contiguously with the sequence of amino acids of the protein. In addition, these immunogenic fragments are typically immunologically reactive with antibodies raised against staphylococcal proteins or with antibodies generated by infection of a mammalian host with Staphylococci or contain T cell epitopes. In one embodiment, the immunogenic fragments also include fragments which, when administered at an effective dose (either alone or as a harbor-bound hapten), elicit a protective immune response against Staphylococcus infection, optionally providing protection against S. aureus and / or S. epidermidis infections Such an immunogenic fragment may include, for example, the protein lacking an N-terminal leader, and / or transmembrane domain and / or C-terminal anchor domain For ClfA, the preferred fragments lack the SD repetition domain towards the C-terminus. ClfA terminal (for example using a fragment in which amino acids 555-927, 556-927, 557-927, 558-927, 559-927 or 560-927 are deleted). For ClfA and alpha toxoid, the preferred fragments have the signal peptide removed to form the mature protein, optionally with an initial methionine residue at the N-terminus to allow recombinant expression.
In one embodiment, immunogenic compositions of the present invention may contain fusion proteins or immunogenic fragments of ClfA. The fusion protein optionally contains heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: β-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), epitopes such as FLAG, myc tag, polyhistidine, or viral surface proteins such as hemagglutinin of the influenza virus, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM 197. The fusion protein may be present in the immunogenic composition of the present invention as a free protein or it may be a saccharide-bound carrier protein.
In one embodiment, the invention also provides an immunogenic fragment of the ClfA protein, i.e., an contiguous portion of the ClfA polypeptide that has the same or substantially the same immunogenic activity as the polypeptide comprising the polypeptide sequence. of SEQ ID NO: 3. In other words, the fragment (if necessary when coupled to a carrier) is capable of inducing an immune response that recognizes the ClfA polypeptide. Such an immunogenic fragment may include, for example, the ClfA polypeptide lacking an N-terminal leader, and / or the SD repetition domain towards the C-terminus of ClfA. In a preferred aspect, the immunogenic fragment of ClfA comprises substantially the entire fibrinogen binding domain and has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity or 100% identity, with the amino acid sequence of any one of SEQ ID NO: 4-12 throughout the length of said sequence.
The fragments may be "autonomous" or comprised within a larger polypeptide of which they form a part or a region, most preferably in the form of a single continuous region within a single larger polypeptide. . Other ClfA fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids derived from the amino acid sequence of SEQ ID NO: 3.
In one embodiment, the ClfA protein is an immunogenic fragment of ClfA comprising the N1 domain, the N2 domain, the N3 domain, the N1 and N2 domains, the N2 and N3 domains or the N1 and N2 and N3 domains. Optionally, the immunogenic fragment of ClfA comprises the N2 and N3 domains and has an amino acid sequence identical to at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% at the sequence of SEQ ID NOS: 6, 7, 11, 12, 15, 16, 17 or 18. Optionally, the immunogenic fragment of ClfA comprises the N1, N2 and N3 domains and has an amino acid sequence identical to at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% to the sequence of SEQ ID NO: 4, 5, 9 or 10.
In one embodiment, the ClfA protein or immunogenic fragment thereof contains an amino acid substitution, deletion or insertion that reduces or abolishes the ability of ClfA to bind to fibrinogen. In one embodiment, the ability of ClfA to bind to fibrinogen is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99%. Such a mutation is typically in the fibrinogen binding region, at the N-terminus of ClfA. The mutation is optionally an amino acid substitution at the level of at least one, two, three or four amino acids Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389, Tyr474, Glu526 or Val527. In one embodiment, the amino acid Pro336 of ClfA is mutated. In one embodiment the Tyr338 amino acid of ClfA is mutated. In one embodiment, Tyr338 is mutated to Ala. In one embodiment, Pro336 and Tyr338 are both mutated, optionally to Alanine or Serine. In one embodiment, ClfA contains two mutations, Pro336 being mutated to Ser and Tyr 338 being mutated to Ala.
In one embodiment, the ClfA protein or immunogenic fragment is present in the immunogenic composition as unconjugated protein. Alternatively, he / she is present in conjugated form with S. aureus Type 5 capsular saccharide or S. aureus type 8 capsular saccharide. In these cases, ClfA can act as a carrier protein and antigen.
In one embodiment, the ClfA protein or immunogenic fragment thereof present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15, 20 to 40 or 30 to 40 μg.
Alpha toxin is an important virulence factor produced by most strains of S. aureus. It is a pore-forming toxin with hemolytic activity. It has been shown that antibodies directed against alpha toxin neutralize the deleterious and lethal effects of alpha toxin in animal models (Adlam et al., 1977 Infect Immun 17, 250). Human platelets, endothelial cells, and mononuclear cells are sensitive to the effects of alpha toxin. For the alpha toxin to be used in an immunogenic composition, it is typically detoxified by chemical treatment or mutation to produce alpha toxoid.
In one embodiment, the immunogenic composition comprises an alpha toxoid. Optionally, the alpha toxoid has an amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1,2, 13 or 14.
The high toxicity of alpha toxin requires that it be detoxified before being used as an immunogen. This can be obtained by chemical treatment, for example by treatment with formaldehyde, glutaraldehyde or other crosslinking reagents or by chemical conjugation with bacterial polysaccharides, as described above.
Another way of eliminating toxicity is to introduce point mutations that eliminate toxicity while maintaining the immunogenicity of the toxin. The introduction of a point mutation at the amino acid level of alpha toxin where a histidine residue is replaced by a leucine residue results in the elimination of toxicity while maintaining immunogenicity (Menzies and Kernodle 1996 Infect Immun 64, 1839). Histidine appears to play a vital role in the proper oligomerization required for pore formation and the mutation of this residue leads to a loss of toxicity. The modification of histidine 35 may be a substitution by Lys, Arg, Ala, Leu or Glu. It is optionally possible to use a point mutation of alpha toxin in Asp24, Lys37, His48, Lys58, Asp100, Ile107, Glu111, Met113, Asp127, Asp128, Gly130, Gly134, His144, Lys147, Gln150, Asp152, Phe153. , Lys154, Val169, Asn173, Arg200, Asn214, Leu219 or His259 to reduce toxicity.
When incorporated into immunogenic compositions of the present invention, the alpha toxoid is optionally detoxified by mutation of His 35, for example by replacing His 35 with Leu or Arg. In an alternative embodiment, the alpha toxoid is detoxified by conjugation to other components of the immunogenic composition, for example S. aureus type 5 polysaccharide and / or S. aureus type 8 polysaccharide. In one embodiment, the alpha toxoid is detoxified by both the introduction of a point mutation and by conjugation to S. aureus Type 5 polysaccharide and / or S. aureus Type 8 polysaccharide.
In one embodiment, the immunogenic composition comprises alpha toxoid that contains a point mutation that decreases the toxicity of the alpha toxin, for example, at the amino acid level 35. The alpha toxoid optionally contains a point mutation at the of the amino acid where the histidine is replaced by an amino acid arginine.
In one embodiment, the alpha toxoid is present in the immunogenic composition as unconjugated protein. Alternatively, alpha toxoid is conjugated to S. aureus Type 5 capsular saccharide and / or S. aureus type 8 capsular saccharide.
In one embodiment, the alpha toxoid is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15, 20 to 40 or 30 to 40 μg. In one embodiment, ClfA and alpha toxoid are present at the same dose in the immunogenic composition. In one embodiment, the saccharide dose of capsular saccharide conjugates of Type 5 and 8 is greater than the dose of ClfA protein and alpha toxoid.
In one embodiment, the immunogenic composition further comprises a S. aureus Type 5 capsular saccharide conjugated to a carrier protein to form a S. aureus Type 5 capsular saccharide conjugate, wherein the capsular saccharide conjugate of Type 5 S. aureus is administered at a saccharide dose of 3 to 50 μg, 3 to 25 μg, 3 to 2 μg, 3 to 12 μg, 5 to 50 μg, 5 to 25 μg, 5 to 20 μg, 5 to 12 μg, at 10 μg, 7 to 2 μg, 7 to 15 μg or 8 to 12 μg.
In one embodiment, the immunogenic composition further comprises a S. aureus Type 8 capsular saccharide conjugated to a carrier protein to form a S. aureus type 8 capsular saccharide conjugate, wherein the capsular saccharide conjugate of Type 8 S. aureus is administered at a saccharide dose of 3 to 5 μg, 3 to 25 μg, 3 to 2 μg, 3 to 12 μg, 5 to 50 μg, 5 to 25 μg, 5 to 2 μg, at 12 μg, 5 to 1 μg, 7 to 2 μg, 7 to 15 μg or 8 to 12 μg.
In one embodiment, the same saccharide dose of S. aureus Type 5 capsular saccharide conjugate and S. aureus type 8 capsular saccharide conjugate is present in the immunogenic composition; for example, a saccharide dose of 4, 5, 6, 7, 8, 9 or 10 μg of both Type 5 and Type 8 conjugates.
Most strains of S. aureus that cause infections in humans contain Type 5 or Type 8 polysaccharides. Approximately 60% of human strains are Type 8 and approximately 30% are Type 5. Jones Carbohydrate Research 340 , 1097-1106 (2005) used NMR spectroscopy to identify capsular polysaccharide structures as follows:
Type 5 -> 4) -p-D-ManNAcA- (1-4) -a-L-FucNAc (30Ac) - (1 - + 3) -p-D-FucNAc- (1 →
Type 8 -> 3) -p-D-ManNAcA (40Ac) - (1 → 3) -α-L-FucNAc (1 → 3) -α-D-FucNAc (1 -
Polysaccharides can be extracted from the appropriate strain of S. aureus by methods well known to those skilled in the art, for example as described in US6294177, WO 11/41003, WO 11/51917 or Infection and Immunity (1990) 58 (7); 2367. For example, ATCC 12902 is a strain of S. aureus Type 5 and ATCC 12605 is a strain of S. aureus Type 8.
The polysaccharides are of native size or alternatively their size can be reduced, for example by microfluidization, ultrasonic radiation or by chemical treatment such as exposure to pH 5.0-3.0. The invention also covers oligosaccharides derived from polysaccharides of Types 5 and 8 of S. aureus. In one embodiment, S. aureus type 5 capsular saccharide has a molecular weight greater than 25kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa or 90kDa or between 25 to 125kDa, 90 and 125kDa, and 100kDa, 35 and 75KDa or 40 and 70kDa. In one embodiment, S. aureus type 8 capsular saccharide has a molecular weight greater than 25kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa or 90kDa or between 25 and 125kDa, 90 and 125kDa, and 100kDa, 35 and 75KDa or 40 and 70kDa.
In one embodiment, the carrier protein to which the Type 5 and / or Type 8 capsular saccharide is conjugated is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM 197, alpha toxoid, ClfA, and exoprotein A of Pseudomonas aeruginosa. In a preferred embodiment, the carrier protein is CRM 197. In another preferred embodiment, alpha toxoid is used as a carrier protein for capsular saccharide Type 5 and ClfA is used as a carrier protein for capsular saccharide. Type 8.
Capsular polysaccharide Type 5 and / or 8 or oligosaccharides included in the immunogenic composition of the present invention are O-acetylated. In one embodiment, the degree of O-acetylation of the Type 5 capsular polysaccharide or oligosaccharide is 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 6090%, 70-90%, 70-80% or 80-90%. In one embodiment, the O-acetylation degree of the Type 8 capsular polysaccharide or oligosaccharide is 10-100%, 20-100%, 30-100%, 40-100%, 50-100% . 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 6090%, 70-90%, 70-80% or 80-90%. In one embodiment, the degree of O-acetylation of Type 5 and Type 8 capsular polysaccharides or oligosaccharides is ΙΟΙ 00%, 20-100%, 30-100%, 40-100%, 50-100%. %, 60-100%, 70-100%, 80-100%, 90100%, 50-90%, 60-90%, 70-90%, 70-80% or 80-90%. In one embodiment, Type 5 and / or Type 8 capsular saccharides are O-acetylated at 80-100% or 100%.
The degree of O-acetylation of the polysaccharide or oligosaccharide can be determined by any method known in the art, for example, by proton NMR (Lemercinier and Jones 1996, Carbohydrate Research 296; 83-96. Jones and Lemercinier 2002, J Pharmaceutical and Biomedical Analysis 30; 1233-1247, WO 05/033148 or WO 00/56357). Another commonly used method is that described by Hestrin (1949) J. Biol. Chem. 180; 249-261 (the Hestrin method is the preferred one).
O-acetyl groups can be removed by hydrolysis, for example by treatment with a base such as anhydrous hydrazine (Konadu et al 1994, Infect Immun 62, 50485054) or by treatment with 0.1 N of NaOH for a period of time. 1 to 8 hours. In order to maintain high levels of O-acetylation on Type 5 and / or 8 polysaccharide or oligosaccharide, treatments that would lead to hydrolysis of O-acetyl groups are minimized. For example, treatments with extreme pH values are minimized.
Among the problems associated with the use of polysaccharides in vaccination, there is the fact that polysaccharides are per se poor immunogens. Strategies that have been designed to overcome this insufficient immunogenicity include binding the polysaccharide to large protein media, which provide assistance to neighboring T cells. In one embodiment, the polysaccharides used in the context of the present invention are linked to a protein carrier that provides assistance to neighboring T cells. Examples of such carriers which can be used for coupling to the polysaccharide or oligosaccharide immunogens include Diphtheria and Tetanus toxoids (DT, DT Crm197 and TT), keyhole limpet hemocyanin (KLH), Pseudomonas exoprotein A aeruginosa (rEPA) and the purified protein derivative of Tuberculin (PPD), Haemophilus influenzae protein D, pneumolysin or fragments of any of the foregoing. Fragments suitable for use include fragments comprising helper T epitopes. In particular, the protein fragment D
rBE will optionally contain the N-terminus 1/3 of the protein. Protein D is an IgD binding protein extracted from Haemophilus influenzae (EP 0 594 610 B1).
A novel carrier protein whose use would be particularly advantageous in the context of a staphylococcal vaccine is staphylococcal alpha toxoid. The native form can be conjugated to a polysaccharide since the conjugation method reduces the toxicity. Optionally, a genetically detoxified alpha toxin such as the His35Leu or His 35 Arg variants are used as carriers because the residual toxicity is lower. Alternatively, the alpha toxin is chemically detoxified by treatment with a crosslinking reagent, formaldehyde or glutaraldehyde. The conjugation process is an alternative chemical treatment that detoxifies the alpha toxin. A genetically detoxified alpha toxin is optionally chemically detoxified, optionally by treatment with one of a crosslinking reagent, formaldehyde or glutaraldehyde to further reduce toxicity.
The polysaccharides may be bound to the carrier protein (s) by any known method (e.g., Likhite, US Patent 4,372,945, Armor et al., US Patent 4,474,757, Anderson et al WO 10/151544). , Berti et al WO 11/138636, and Jennings et al., US Patent 4,356,170). Optionally, a CDAP conjugation chemistry is performed (see WO 95/08348, WO 07/113222).
In CDAP, the cyanylant 1-cyano-dimethylaminopyridinium tetrafluoroborate reagent (CDAP) is optionally used for the synthesis of polysaccharide-protein conjugates. The cyanilation reaction can be carried out under relatively mild conditions, which avoids the hydrolysis of the alkali sensitive polysaccharides. This synthesis allows a direct coupling to a carrier protein.
The polysaccharide may be solubilized in water or saline. The CDAP can be dissolved in acetonitrile and added immediately to the polysaccharide solution. The CDAP reacts with the hydroxyl groups of the polysaccharide to form a cyanate ester. After the activation step, the carrier protein is added. Amino groups of lysine react with the activated polysaccharide to form a covalent isourea bond. After the coupling reaction, a large excess of glycine is then added to deactivate the residual activated functional groups. The product is then passed through a gel filtration column to remove unreacted carrier protein and residual reagents.
In one embodiment, S. aureus Type 5 capsular saccharide and / or S. aureus Type 8 capsular saccharide is directly conjugated to the protein.
dBE carrier. However, the invention also encompasses conjugates wherein Type 5 and / or 8 capsular saccharides are conjugated via a linker, for example an ADH linker.
In one embodiment, S. aureus type 5 capsular saccharide and / or S. aureus Type 8 capsular saccharide is conjugated by means of a cyanylating reagent, for example CDAP. Alternatively, other conjugation methods such as reductive amination or carbodiimide chemistry (eg EDAC) may be used.
In one embodiment, the ratio of polysaccharide to protein in S. aureus Type 5 capsular saccharide conjugate is between 1: 5 and 5: 1 (w / w), 1: 1 and 1: 5 ( w / w), 1: 2 and 1: 5 (w / w), 1: 3 and 1: 5 (w / w), 1: 2 and 2: 1 (w / w) or 1: 1 and 1: 2 (wt / wt). In one embodiment, the ratio of polysaccharide to protein in S. aureus type 8 capsular saccharide conjugate is between 1: 5 and 5: 1 (w / w), 1: 1 and 1: 5 ( w / w), 1: 2 and 1: 5 (w / w), 1: 3 and 1: 5 (w / w), 1: 2 and 2: 1 (w / w) or 1: 1 and 1: 2 (wt / wt).
In one embodiment, the immunogenic composition of the present invention is mixed with a pharmaceutically acceptable excipient, and optionally an adjuvant to form an immunogenic composition or vaccine.
In one embodiment, the pharmaceutically acceptable excipient is buffered at pH5.0-pH8.0, pH5.5-pH7.0, pH5.0-pH6.5, pH6.5-pH7.0, preferably at pH6 , 0-pH 7.0 or more preferably pH6.0-pH6.5. In one embodiment, the buffering takes place through a buffer with a pKa of 5.0-7.0. In one embodiment, the pharmaceutically acceptable excipient comprises a histidine buffer or a succinate buffer, optionally present at a concentration of 5mM-50mM, or preferably 5mM-15mM.
In one embodiment, the pharmaceutically acceptable excipient comprises NaCl, optionally at a concentration of 50-150mM NaCl, 50-100mM or 140160mM.
The vaccines of the present invention may be adjuvanted, particularly when intended for use in elderly, immunocompromised or chronically ill populations (such as diabetes, end-stage nephropathies or other populations at high risk of staphylococcal infection). ), but also for use in infant populations. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be other metal salts such as calcium, magnesium, iron or zinc. Oil-in-water emulsions, for example comprising metabolizable oil (eg squalene), an emulsifying agent (for example polyoxyethylene sorbitan monooleate) and optionally a tocol (for example, alpha-tocopherol) are also (WO 09/95453).
It is preferred that the adjuvant is selected to be a preferential inducer of a TH1 type response. These high levels of Th1-type cytokines tend to promote the induction of cell-mediated immune responses to a given antigen, whereas high levels of Th2-type cytokines tend to promote the induction of humoral immune responses to the antigen.
The distinction of the Th1 and Th2 type immune responses is not absolute. In reality, an individual will support an immune response that is described as being predominantly Th1 or Th2 predominantly. However, it is often convenient to consider the cytokine families in terms of that described in murine CD4 + ve T cell clones by Mosmann and Coffman (Mosmann, TR and Coffman, RL (1989) TH1 and TH2 cells: Different patterns of Lymphokine Secretion Lead to Different Functional Properties (Annual Review of Immunology, 7, p145-173) Traditionally, Th1-type responses are associated with the production of INF-gamma and IL-2 cytokines by T-lymphocytes. Cytokines often directly associated with the induction of Th1-type immune responses are not produced by T cells, such as IL-12 In contrast, Th2-like responses are associated with secretion of II-4, IL-5 IL-6, IL-10 Appropriate adjuvant systems that promote a predominant Th1 response include: monophosphoryl lipid A or a derivative thereof (or detoxified lipid A in general - see for example WO2005107798) ), especially 3-de-O-acylated monophosphoryl A lipid (3D-MPL) (for its preparation, cf. GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid, with either an aluminum salt (e.g., aluminum phosphate or aluminum hydroxide), or an emulsion oil in the water. In these combinations, the antigen and 3D-MPL are contained in the same particle structures, allowing a more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further increase the immunogenicity of an antigen adsorbed on alum [Thoelen et al. Vaccine (1998) 16: 708-14; EP 689454-B1],
Another system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is deactivated. with cholesterol as described in WO 96/33739. Another adjuvant formulation using QS21, 3D-MPL and tocopherol in oil-in-water emulsion is described in WO 95/17210. In one embodiment, the immunogenic composition further comprises a saponin, which may be QS21. The formulation may also include an oil-in-water emulsion and tocopherol (WO 95/17210). Oligonucleotides containing unmethylated CpG (WO 96/02555) and other immunomodulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of a TH1 response and are suitable for use in the context of the present invention.
However, the inventors have found that in a clinical trial, the addition of an oil-in-water emulsion adjuvant did not produce an increase in immunogenicity. In view of the increased reactogenicity that may be associated with the use of an adjuvant, an embodiment of the present invention uses a non-adjuvanted immunogenic composition, for example an immunogenic composition in which none of the staphylococcal components present are adsorbed. an adjuvant or an immunogenic composition in which the staphylococcal components are not mixed with an oil-in-water emulsion adjuvant. Staphylococcal components include 1, 2, 3 or 4 of S. aureus Type 5 capsular saccharide conjugate, S. aureus Type 8 capsular saccharide conjugate, a ClfA protein or an immunogenic fragment thereof. an alpha toxoid.
Another aspect of the present invention is a vaccine comprising the immunogenic composition described above and a pharmaceutically acceptable excipient. The vaccine preparations of the present invention can be used to protect or treat a human subject to S. aureus infection by administering said vaccine systemically or mucosally. These administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or via oral mucosa / food, respiratory, genitourinary administration.
Vaccine preparation is generally described in Vaccine Design ("The Subunit and Adjuvant Approach" (Eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). The encapsulation within liposomes is described by Fullerton, US Patent 4,235,877.
Vaccines of the present invention may be stored in solution or lyophilized. Optionally the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. It is typical that they are lyophilized and reconstituted extemporaneously before use. Lyophilization may result in a more stable composition (vaccine). The invention also encompasses the method of making the immunogenic compositions and vaccines of the present invention. In one embodiment, the method of the present invention is a method for making a vaccine comprising the steps of conjugating a S. aureus type 5 capsular saccharide to a carrier protein to form a type 5 capsular saccharide conjugate. S. aureus, b) conjugate a S. aureus Type 8 capsular saccharide conjugated to a carrier protein to form a S. aureus type 8 capsular saccharide conjugate, and c) combine the type 5 capsular saccharide conjugate of S. aureus, S. aureus Type 8 capsular saccharide conjugate, a ClfA protein or an immunogenic fragment thereof, and an alpha toxoid to form the immunogenic composition. In one embodiment, the method comprises another step of adding a pharmaceutically acceptable excipient. The invention also encompasses a method of treating staphylococcal infections, particularly nosocomial hospital infections. The use of this immunogenic composition or vaccine of the present invention is particularly advantageous in cases of elective surgery, particularly when the subjects are immunized with a single dose. These patients know the date of the procedure in advance and can advantageously be inoculated in advance. In one embodiment, the subject is immunized with a single dose of the immunogenic composition of the present invention from 5 to 60, 6 to 40, 7 to 30 or 7 to 15 days prior to admission to hospital. In one embodiment, the subject is immunized with a single dose of the immunogenic composition of the present invention from 5 to 60, from 6 to 40, from 7 to 30 or 7 to 15 days prior to a planned intervention in the hospital. for example a surgical procedure such as cardio-thoracic surgery. Typically, adults over 16 years of age waiting for elective surgery are treated with the immunogenic compositions and vaccines of the present invention. Alternatively, children aged 3 to 16 years waiting for elective surgery are treated with the immunogenic compositions and vaccines of the present invention.
It is also possible to inoculate the vaccine of the present invention with health personnel.
The vaccine preparations of the present invention can be used to protect or treat a human subject to S. aureus infection by administering said vaccine systemically or mucosally. These administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or via oral mucosa / food, respiratory, genitourinary administration.
One embodiment of the present invention is a method for preventing or treating infections or staphylococcal diseases, comprising the step of administering the immunogenic composition or vaccine of the present invention to a patient in need thereof.
Another embodiment of the present invention is a use of the immunogenic composition of the present invention in the manufacture of a vaccine for the treatment or prevention of staphylococcal infections or diseases, optionally post-surgical staphylococcal infections.
Vaccine preparation is generally described in Vaccine Design ("The Subunit and Adjuvant Approach" (Eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). The encapsulation within liposomes is described by Fullerton, US Patent 4,235,877.
The terms "comprising", "understand" and "includes" herein are intended by the inventors to be substituted by the terms "consisting of", "consisting of" and "consisting of" respectively, in all cases.
All references to or patent applications mentioned in this specification are incorporated by reference.
The following examples are intended to clarify the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.
Examples
Example 1 Protein Sequences SEQ ID NO: 1
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSF IDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTK EYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQN WGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNID VIYERVRDDYQLHWTSTNWKGTNTKDKWIDRS SERYKIDWEKEEMTN SEQ ID NO: 2
MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGL IGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGS MKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTK DKWIDRS SERYKIDWEKEEMTN SEQ ID NO: 3
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSD
TKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETT
SNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADA
PVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG
DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF
YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV
NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN
EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPEDSDSDPGSDSGSDSNSDSGSDSGSDSTSDSGSDSASDSDSA
SDSDSASDSDSASDSDSASDSDSDNDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS
DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD
SDSDSDSDSDSDSDSDSASDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSESDSDSDSDSDSDSDSDS
DSDSDSASDSDSGSDSDSSSDSDSESDSNSDSESVSNNNVVPPNSPKNGTNASNKNEAKDSKEPLPDTGSEDEAN
TSLIWGLLASIGSLLLFRRKKENKDKK SEQ ID NO: 4
MSENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTT
TNQANTPATTQSSNTNAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQ
AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNG
VTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYE
KYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPE
NFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSG
DGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 5 MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKTDDTNViBËë
TKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETT
SNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADA
PVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG
DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF
YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV
NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN
EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 6
SLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTST
AKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVL
VDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNA
ADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNI
IWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 7
GTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG
DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF
YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV
NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN
EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 8
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSD TKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETT SNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDWNQAVNTSAPRMRAFSLAAVAADA PVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPEDSDSDPGSDSGSDSNSDSGSDSGSDSTSDSGSDSASDSDSA SDSDSASDSDSASDSDSASDSDSDNDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD S DS DS DS DS DS DS DS DSAW DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS FSD S DS DS DS DS DS DS DS DS DSDSDSASDSDSGSDSDSSSDSDSESDSNSDSESVSNNNVVPPNSPKNGTNASNKNEAKDSKEPLPDTGSEDEAN TSLIWGLLASIGSLLLFRRKKENKDKK SEQ ID NO: 9
MSENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTT TNQANTPATTQSSNTNAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQ AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNG VTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYE KYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPE NFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSG DGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 10 MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVâÆi
TKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETT
SNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADA
PVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG
DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF
YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV
NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN
EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQID NO: 11
SLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTST
AKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVL
VDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNA
ADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIWVNGHIDPNSKGDLALRSTLYGYNSNI
IWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQID NO: 12
GTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAG
DQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKF
YNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFV
NPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDN
EVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQID NO: 13
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMRKKVFYSF
IDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTK
EYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQN
WGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNID
VIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQID NO: 14
MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMRKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGL IGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGS MKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTK DKWIDRS SERYKIDWEKEEMTN SEQ ID NO: 15
MASLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVT
STAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKT
VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVD
NAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS
NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 16
MAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMA
GDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGK
FYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYF
VNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWD
NEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQID NO: 17
MASLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVT
STAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKT
VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVD
NAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS
NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQID NO: 18
MAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGD
QVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFY
NLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVN
PENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIWVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNE
VAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE
Example 2 Preparation of vaccine components
A four-component staphylococcal vaccine was prepared containing S. aureus Type 5 capsular polysaccharide conjugated to a tetanus toxoid-carrying protein, S. aureus type 8 capsular polysaccharide conjugated to a carrier protein of S. aureus. tetanus toxoid, a ClfA fragment containing the N2 and N3 domains and point mutations at residues 336 and 338, wherein P336 is changed to serine and Y338 is changed to alanine, and alpha toxoid which is detoxified by a point mutation at residue 35, with H35 being changed to arginine. Capsular polysaccharides were conjugated to tetanus toxoid using CDAP as a coupling agent. This conjugation method is described in WO 07/113222.
Four formulations of the staphylococcal vaccine were carried out: 5/10 containing: 5 μg of Type 5 conjugate saccharide-tetanus toxoid, 5 μg of Type 8 conjugate saccharide-tetanus toxoid, 10 μg of alpha toxoid and 10 μg of tetanus toxoid Truncated ClfA described above. 10/30 containing: 10 μg of saccharide dose of Type 5 conjugate - tetanus toxoid, 10 μg of Type 8 conjugate saccharide dose - tetanus toxoid, 30 μg of alpha toxoid and 30 μg of the truncated ClfA described above. 5 / 10OAS containing: 5 μg of saccharide dose of Type 5 conjugate - tetanus toxoid, 5 μg of saccharide dose of Type 8 conjugate - tetanus toxoid, 10 μg of alpha toxoid and 10 μg of the truncated ClfA described above, with adjuvant uRP oil-in-water emulsion containing squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate. 10 / 30AS containing: 10 μg Saccharide Dose of Type 5 Conjugate - Tetanus Toxoid, 10 μg of Saccharide Dose of Type 8 Conjugate - Tetanus Toxoid, 30 μg of Alpha Toxoid and 30 μg of the Truncated ClfA described above, with adjuvant an oil-in-water emulsion containing squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate.
Example 3 Results of Clinical Trials with Staphylococcal 4-Component Vaccine
A phase I clinical trial was conducted with a total of 88 healthy adults aged 18 to 40 years old. The control group contained 30 subjects who were inoculated with saline. The remaining subjects were divided into four arms, 15/14 subjects being immunized with each of the formulations described in Example 2 (5/10, 5 / 10AS, 10/30 and 10 / 30AS). Vaccine doses were administered at the beginning of the trial and after one month and six months. Blood samples were taken for humoral analysis at day 0, 7, 14 and 30 after each dose and at day 360 and 540.
The details of the topics are provided below.
Group N Average age% females 5/10 15 31.1 73.3 5 / 10AS 15 31.9 33.3 10/30 14 30.9 42.9 10 / 30AS 14 30.6 50
Saline 30 30.1 50 Reactogenicity and safety
The 4-component staphylococcal vaccine was generally safe and well tolerated. No serious adverse events and no potential immunological disturbances were observed after the first and second doses. The percentage of subjects reporting pain, redness and swelling after dose 1 and dose 2 is shown in Figures 1 to 3. Pain was reported at the injection site in 78.6 to 100 % of subjects in the vaccinated groups compared to 3 to 4% in the control group (see Figure 1). Only one level 3 case has been reported. The results concerning the incidence of redness and swelling appear in Figures 2 and 3. For both parameters, a stronger tendency to the incidence of redness / swelling was observed following the administration of the second dose in comparison observed after a single dose for the 10/30 arm of the study.
immunogenicity
Blood samples taken on subjects at day 0 and 7, 14 and 30 days after the first, second and third immunizations were tested by Luminex or ELISA to establish the level of IgG produced against each antigen of the four-component staphylococcal vaccine.
The results relating to immunogenicity appear in Figures 4 to 8 and in Tables 1 to 5 below.
In prevaccination, seropositivity of 83.3 to 100% was recorded for all trials. Despite considerable levels of general immunity, the 4-component vaccine was able to elicit a robust immune response against the 4 components.
Figures 4 to 7 show that for CPS5, CPS8, alpha toxoid and ClfA, the first immunization produced the greatest increase in immunogenicity, with large increases in GMC occurring on days 14 and 30. The second immunization at Day 30 did not result in a further increase in immunogenicity and GMC levels remained at a similar level between days 30 and 60. Figure 8 shows that the third immunization after 6 months did not induce further increase in GMC with GMC levels that remain approximately the same for all four components between day 30 and day 540. A single immunization is therefore an effective means of producing a maximal immune response.
The immunogenicity results obtained with the 10/30 assay appear to be more satisfactory than those obtained with the 5/10 assay, with an approximate increase in GMC of 1-5-2 fold for CPS5, CPS8 and alpha toxoid. In the case of ClfA, the increase in GMC was approximately 3.8 times at the maximum dose. Addition of the oil emulsion adjuvant in water did not increase the immunogenicity of the 4-component vaccine as demonstrated by a comparison of antibody response elicited by the 5/10 and 5/10 arms and 10/30 and 10 / 30AS arms.
Table 1 Seropositivity and GMC for Ab.IgG CPS 5 antibodies of Stapëi aureus. (protocol compliant cohort for immunogenicity analysis)
5/10 = 5pg of CPS5-TT, 5pg of CPS8-TT, 10pg of ClfA, 10pg of toxoid at 5 / 10OAS = 5pg of CPS5-TT, 5pg of CPS8-TT, 10pg of ClfA, 10pg of toxoid a with AS03B as a 10/30 adjuvant = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid at 10/30 μS = 1 g of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid a with AS03B as adjuvant SALINE = grouping of SALINE1 and SALINE2 GMC = geometric mean concentration of antibodies calculated on all subjects N = number of subjects for which results are available n /% = number / percentage of subjects whose concentration is within the specified range 95% Cl = 95% confidence interval; U = Lower Limit, LS = Upper Limit PRE = Pre-dose 1 PI (J7) = 7 days after dose 1 PI (J14) = 14 days after dose 1 PI (J30) = 30 days after dose 1 (sampling blood flow at Visits 5 or 6) PII (J37) = 7 days after dose 2 PII (J44) = 14 days after dose 2 PII (J60) = 30 days after dose 2
Table 1 Seropositivity and GMC for Ab.IgG CPS 8 antibodies from Statflue aureus. (protocol compliant cohort for immunogenicity analysis)
5/10 = 5μ9 of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid at 5 μSOAS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μl of ClfA, 10 μl of toxoid a. with AS03B as adjuvant 10/30 = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30 μg of ClfA, 3 μg of toxoid at 10 / 30AS = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30μ9 of ClfA , 30μ9 of toxoid a with AS03B as adjuvant SALINE = grouping of SALINE1 and SALINE2 GMC = geometric mean concentration of antibodies calculated on all subjects N = number of subjects for which results are available n /% = number / percentage of subjects of which the concentration is within the specified range 95% Cl = 95% confidence interval; U = Lower Limit, LS = Upper Limit PRE = Pre-dose 1 PI (J7) = 7 days after dose 1 PI (J14) = 14 days after dose 1 PI (J30) = 30 days after dose 1 (sampling blood flow at Visits 5 or 6) PII (J37) = 7 days after dose 2 PII (J44) = 14 days after dose 2 PII (J60) = 30 days after dose 2
Table 2 Seropositivity and GMC for Ab.IgG alpha-toxin Staph aureus (protocol compliant cohort for immunogenicity analysis)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid at 5 μS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μ μ of a toxin with AS03B as adjuvant 10/30 = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30μg of ClfA, 30μ9 of toxoid at 10 / 30AS = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30μ9 of ClfA, 30μ9 of anatoxin a with AS03B as adjuvant SALINE = grouping of SALINE1 and SALINE2 GMC = geometric mean concentration of antibodies calculated on all subjects N = number of subjects for which results are available n /% = number / percentage of subjects whose concentration is within the specified range 95% Cl = 95% confidence interval; U = Lower Limit, LS = Upper Limit PRE = Pre-dose 1 PI (J7) = 7 days after dose 1 PI (J14) = 14 days after dose 1 PI (J30) = 30 days after dose 1 (sampling blood flow at Visits 5 or 6) PII (J37) = 7 days after dose 2 PII (J44) = 14 days after dose 2 PII (J60) = 30 days after dose 2
Table 4 Seropositivity and GMC for Ab.IgG ClfA antibodies from StajS aureus (protocol compliant cohort for immunogenicity analysis)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid at 5 μSO = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 4 μg of ClfA, 10 μg of toxoid a with AS03B as adjuvant 10/30 = 10pg of CPS5-TT, 10pg of CPS8-TT, 30pg of ClfA, 30μg of toxoid at 10 / 30AS = 10pg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30pg of toxoid a with AS03B as adjuvant SALINE = grouping of SALINE1 and SALINE2 GMC = geometric average concentration of antibodies calculated on all subjects N = number of subjects for which results are available n /% = number / percentage of subjects whose concentration is within the specified range 95% Cl = 95% confidence interval; U = Lower Limit, LS = Upper Limit PRE = Pre-dose 1 PI (J7) = 7 days after dose 1 PI (J14) = 14 days after dose 1 PI (J30) = 30 days after dose 1 (sampling blood flow at Visits 5 or 6) PII (J37) = 7 days after dose 2 PII (J44) = 14 days after dose 2 PII (J60) = 30 days after dose 2
Table 5 Seropositivity and GMC for Ab.IgG Tox C toxins (protocol compliant cohort for immunogenicity analysis)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid at 5 μSOAS = 5 μg of CPS5-TT, 5 μ9 of CPS8-TT, 10 μg of ClfA, 10 μl of toxoid a. with AS03B as adjuvant 10/30 = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30μ9 of ClfA, 30μ9 of toxoid at 10 / 30AS = 10μ9 of CPS5-TT, 10μ9 of CPS8-TT, 30μ9 of ClfA, 30μ9 of anatoxin a with AS03B as adjuvant SALINE = grouping of SALINE1 and SALINE2 GMC = geometric mean concentration of antibodies calculated on all subjects N = number of subjects for which results are available n /% = number / percentage of subjects whose concentration is within the specified range 95% Cl = 95% confidence interval; Ll = Lower Limit, LS = Upper Limit PRE = Prior to 1 PI dose (J7) = 7 days after 1 PI dose (J14) = 14 days after 1 PI dose (J30) = 30 days after dose 1 (withdrawal) blood flow at Visits 5 or 6) PII (J37) = 7 days after dose 2 PII (J44) = 14 days after dose 2 PII (J60) = 30 days after dose 2

权利要求:
Claims (42)
[1]
REVENDICATIONS
1. Composition immunogène comprenant : (i) une protéine ClfA de Staphylococcus aureus ou un fragment immunogène de celle-ci à une dose de 5 à 50, 10 à 30, 5 à 15 ou 20 à 40 pg, et (ii) une anotoxine alpha qui contient une mutation ponctuelle qui diminue la toxicité de l’anatoxine alpha à une dose de 5 à 50, 10 à 30, 5 à 15 ou 20 à 40 pg et (iii) un excipient pharmaceutiquement acceptable ; dans laquelle le pH de la composition immunogène est un pH de 5,0 à 8,0, destinée à être utilisée dans le traitement ou la prévention des infections à Staphylococcus aureus où un patient humain est immunisé avec une dose unique de la composition immunogène.
[2] 2. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 1, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est identique à au moins 90 % à la séquence polypeptidique de l’un quelconque de SEQ ID N° : 3 à 12 ou 15 à 18 sur toute leur longueur.
[3] 3. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 1 ou 2, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est un fragment de ClfA comprenant un domaine N2.
[4] 4. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon les revendications 1 à 3, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est un fragment de ClfA comprenant un domaine N3.
[5] 5. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 4, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est un fragment de ClfA comprenant un domaine N1.
[6] 6. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 5, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci comprend les domaines N2-N3 et a une séquence polypeptidique identique à au moins 90 % à la séquence de SEQ ID N° : 6, 7, 11, 12, 15, 16, 17 ou 18.
[7] 7. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 6, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci contient une substitution d’acide aminé qui réduit la capacité de ClfA à se lier au fibrinogène.
[8] 8. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 7, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci contient une substitution d’acide aminé au niveau d’a§E' moins un des acides aminés Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389, Tyr474, Glu526 ou Val527.
[9] 9. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 7, dans laquelle l’acide aminé Pro336 est muté en Ser et/ou Y338 est muté en Ala.
[10] 10. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 9, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est présent(e) dans la composition immunogène sous forme de protéine non conjuguée.
[11] 11. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 10, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est conjugué(e) au saccharide capsulaire de Type 5 de S. aureus.
[12] 12. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 11, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est conjugué(e) au saccharide capsulaire de Type 8 de S. aureus.
[13] 13. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 12, dans laquelle la protéine ClfA ou le fragment immunogène de celle-ci est présent(e) dans la composition immunogène à une dose de 20 à 40 !g.
[14] 14. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 1, dans laquelle l’anatoxine alpha a une séquence d’acides aminés identique à au moins 90 % à SEQ ID N° : 1,2, 13 ou 14.
[15] 15. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 1, dans laquelle l’anatoxine alpha contient une mutation ponctuelle au niveau de l’acide aminé 35.
[16] 16. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 15, dans laquelle la mutation ponctuelle au niveau de l’acide aminé 35 remplace une histidine par un acide aminé arginine.
[17] 17. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 16, dans laquelle l’anatoxine alpha est présente dans la composition immunogène sous forme de protéi^ê' non conjuguée.
[18] 18. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 17, dans laquelle l’anatoxine alpha est conjuguée au saccharide capsulaire de Type 5 de S. aureus.
[19] 19. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 18, dans laquelle l’anatoxine alpha est conjuguée au saccharide capsulaire de Type 8 de S. aureus.
[20] 20. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 19, dans laquelle ClfA et l’anatoxine alpha sont présents à la même dose dans la composition immunogène.
[21] 21. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 20, dans laquelle la composition immunogène comprend un saccharide capsulaire de Type 5 de S. aureus conjugué à une protéine porteuse pour former un conjugué de saccharide capsulaire de Type 5 de S. aureus, dans laquelle le conjugué de saccharide capsulaire de Type 5 de S. aureus est administré à une dose de saccharide
[22] 22. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 21, dans laquelle la composition immunogène comprend un saccharide capsulaire de Type 8 de S. aureus conjugué à une protéine porteuse pour former un conjugué de saccharide capsulaire de Type 8 de S. aureus, dans laquelle le conjugué de saccharide capsulaire de Type 8 de S. aureus est administré à une dose de saccharide de 3 à 50pg, 5 à 25pg, 3 à 20pg, 3 à 12pg, 5 à 10pg, 7 à 20pg, 7 à 15pg ou 8 à 12pg.
[23] 23. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 21 ou 22, dans laquelle le saccharide capsulaire de Type 5 de S. aureus a un poids moléculaire supérieur à 25kDa, 40kDa ou 50kDa ou compris entre 25 et 300kDa, 50 et 250kDa, 70 et 150kDa, 25 et 125kDa, 90 et 125kDa, 30 et 100kDa, 35 et 75KDa ou 40 et 70kDa.
[24] 24. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 23, dans laquelle le saccharide capsulaire de Type 8 de S. aureus a un poids moléculaire supérieur à 25kDa, 40kDa ou 50kDa ou compris entre 25 et 300kDa, 50 et 250kDa, 70 et 150kDa, 25 et 125kDa, 90 et 125kDa, 30 et 100kDa, 35 et 75KDa ou 40 et 70kDa.
[25] 25. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 24, dans laquelle la protéine porteuse à laquelle le saccharide capsulaire de Type 5 est conjugué est sélectionnée parmi le groupe composé de l’anatoxine tétanique, l’anatoxine diphthérique, CRM197, l’anatoxine alpha, ClfA, l’exoprotéine A de Pseudomonas aeruginosa.
[26] 26. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 25, dans laquelle la protéine porteuse à laquelle le saccharide capsulaire de Type 8 est conjugué est sélectionnée parmi le groupe composé de l’anatoxine tétanique, l’anatoxine diphthérique, CRM197, l’anatoxine alpha, ClfA et l’exoprotéine A de Pseudomonas aeruginosa.
[27] 27. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 25 ou 26, dans laquelle la protéine porteuse est l’anatoxine tétanique ou CRM197.
[28] 28. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 27, dans laquelle le saccharide capsulaire de Type 5 de S. aureus et/ou le saccharide capsulaire de Type 8 de S. aureus est O-acétylé à 50-100 % ou à 75-100 %.
[29] 29. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 28, dans laquelle le saccharide capsulaire de Type 5 de S. aureus et/ou le saccharide capsulaire de Type 8 de S. aureus est directement conjugué à la protéine porteuse.
[30] 30. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 29, dans laquelle le saccharide capsulaire de Type 5 de S. aureus est conjugué au moyen d’un réactif cyanylant.
[31] 31. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 30, dans laquelle le saccharide capsulaire de Type 8 de S. aureus est conjugué au moyen d’un réactif cyanylant.
[32] 32. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 30 ou 31, dans laquelle le réactif cyanylant est du CDAP.
[33] 33. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 32, dans laquelle le rapport du polysaccharide sur la protéine dans le conjugué de saccharide capsulai de Type 5 de S. aureus est compris entre 1:5 et 5:1 (pds/pds) ou entre 1:2 et 2:1 (pds/pds), 1:2 à 1:5 (pds/pds) ou 1:1 et 1:2 (pds/pds).
[34] 34. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 33, dans laquelle le rapport du polysaccharide sur la protéine dans le conjugué de saccharide capsulaire de Type 8 de S. aureus est compris entre 1:5 et 5:1 (pds/pds) ou entre 1:2 et 2:1 (pds/pds), 1:2 à 1:5 (pds/pds) ou 1:1 et 1:2 (pds/pds).
[35] 35. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 34, dans laquelle la même dose de saccharide de saccharide capsulaire de Type 5 de S. aureus et de saccharide capsulaire de Type 8 de S. aureus est présente dans la composition immunogène.
[36] 36. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 21 à 35, dans laquelle le pH de la composition immunogène est un pH 6,0 à pH 7,0.
[37] 37. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 36, dans laquelle l’excipient pharmaceutiquement acceptable comprend un tampon histidine ou succinate.
[38] 38. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 37, dans laquelle l’excipient pharmaceutiquement acceptable comprend du NaCl.
[39] 39. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon la revendication 38, dans laquelle l’excipient pharmaceutiquement acceptable comprend 50 à 150mM de NaCl.
[40] 40. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 39, dans laquelle la composition immunogène ne contient pas une émulsion huile dans l’eau.
[41] 41. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications1 à 40, dans laquelle la composition immunogène est sans adjuvant.
[42] 42. Composition immunogène destinée à être utilisée dans le traitement ou la prévention des infections à S. aureus selon l’une quelconque des revendications 1 à 41, dans laquelle la dose unique de la composition immunogène est administrée de 5 à 60, de 6 à 40, de 7¾ 30 ou de 7 à 15 jours avant une intervention prévue à l’hôpital.

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AU2018290298A2|2020-02-27|Immunogenic compositions

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WO2017011338A1|2017-01-19|Sortase-mediated coupling of immunogenic polysaccharide-protein conjugates and their use

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