IMMUNOGENIC COMPOSITION FOR USE IN THERAPY

专利摘要:
The application describes a method of immunizing against infection by Staphylococcus aureus comprising a step of administering to a human patient a single dose of an immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a transport protein for forming a S. aureus Type 5 capsular saccharide conjugate, wherein the S. aureus Type 5 capsular saccharide conjugate is administered at a saccharide dose of 3 to 50 µg.
公开号:BE1021938B1
申请号:E2014/0416
申请日:2014-06-03
公开日:2016-01-27
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:

IMMUNOGENIC COMPOSITION FOR USE IN THERAPY Technical field
The present invention relates to the field of immunogenic compositions and vaccines against staphylococci, the manufacture and use of such compositions in medicine. More particularly, it relates to the use of conjugates consisting of a capsular saccharide derived from S. aureus, conjugated to a transport protein. Such conjugates can be combined with selected staphylococcal protein antigens to form multivalent compositions.
Context
Staphylococcus aureus (S. aureus) is a commensal Gram positive bacterium that colonizes nostrils, axillary cavities, pharynx, and other mucosal and cutaneous surfaces of approximately 30% of human subjects. It is estimated that S. aureus is responsible for 20 to 25% of all nosocomial infections (Wisplinghoff et al., Clin Infect Dis., 2004; 39; 309-317), resulting in a hospital stay three times longer and a 5-fold increase in hospital deaths for infected patients compared to patients without such infections (Noskin et al., Intern Med 2005; 165; 1756-1761). S. aureus infections can be associated with hospital mortality rates as high as 25%. From the historical point of view, S. aureus has been associated mainly with nosocomial infections. The severity of such infections has increased with the very large recent increase in S. aureus infection associated with antibiotic resistance.
Staphylococcus aureus is the most common cause of nosocomial infections with significant 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 to staphylococcal antigens was investigated (WO 00/15238, WO 00/12132) as well as immunotherapy using a monoclonal antibody against lipoteichoic acid (WO 98/57994) . So far, however, no license has been granted for use. Several immunotherapy candidates have failed to show efficacy in humans. These include: Altastaph (Nabi Biopharmaceuticals) containing the purified CP5 and CP8 antibodies from subjects vaccinated with StaphVAX ™ (study vaccine developed and marketed by Nabi Biopharmaceuticals, Rockville, MD, USA); Veronate (Inhibitex), polyclonal antibodies targeting S. aureus agglutination factor A (ClfA) and S. epidermidis SdrG adhesion protein; Aurexis (Tefibazumab, Inhibitex), monoclonal antibodies targeting ClfA; Aurograb (NeuTec Pharma), a single-chain antibody against an ATP-binding cassette transporter; and Pagibaximab (Biosynexus), a monoclonal anti-lipoteichoic acid antibody (Dejonge et al., J. Paediatrics 2007; 151; 260-265; Rupp et al., Antimicrob, Agents Chemother., 2007; 51; 4249-4254).
An alternative approach would be the use of active vaccination to generate a polyclonal immune response against staphylococci. One approach reported in WO 03/61558 utilizes S. aureus Type 5 and Type 8 capsular polysaccharide conjugates conjugated to Pseudomonas exoprotein A (StaphVAX - Nabi Biopharmaceuticals). Another approach used protein
IsdB of S. aureus (V710 - Merck & Co) but failed to demonstrate efficacy (Fowler et al 2013, JAMA 309, 1368-1378).
There are many problems associated with the development of a vaccine against S. aureus infection. The failure of single-component vaccines (capsular polysaccharide or IsdB protein) suggests that a more complex vaccine containing multiple components may be required to induce protective immunity. However, the combination of different antigens in an immunogenic composition can lead to interference occurring 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 simple. There remains a need to develop an effective vaccine against staphylococcal infection, especially in view of the increased frequency of multidrug-resistant strains.
In the case of immunization against nosocomial staphylococcal infection, immunization can often take place only briefly before hospitalization or surgery or the installation of an indwelling catheter. Therefore, it would be advantageous to obtain high levels of immunity with a single immunization. The use of lower doses of conjugate also has advantages of relative vaccine production efficiency and associated economic benefits.
Therefore, there is provided a method of immunizing against Staphylococcus aureus infection comprising a step of administering to a human patient a single dose of an immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a protein to form a capsular saccharide conjugate of S. aureus Type 5, wherein the capsular saccharide conjugate of S. aureus Type 5 is administered at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg 3 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
In a second aspect of the invention, there is provided an immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a transport protein to form a capsular saccharide conjugate of S. aureus Type 5, wherein the capsular saccharide conjugate S. aureus Type 5 is administered at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 μg. at 12 μg, for use in the treatment or prevention of Staphylococcus aureus infection where a single dose of the immunogenic composition is administered to a human patient.
In a third aspect of the invention, there is provided an immunogenic composition comprising a capsular saccharide of S. aureus Type 5 conjugated to a transport protein, a capsular saccharide of 5. aureus Type 8 conjugated to a transport protein, a protein ClfA or one of its fragments and an alpha toxoid.
In a fourth aspect of the invention, there is provided a vaccine against a S. aureus Type 5 capsular saccharide conjugated to a transport protein, a capsular saccharide of S. aureus Type 8 conjugated to a transport protein, a ClfA protein. or one of its fragments and an alpha toxoid and a pharmaceutically acceptable excipient.
In a fifth aspect of the invention there is provided a method of making the immunogenic composition or vaccine of the invention comprising the steps of a) conjugating a capsular saccharide of S. aureus Type 5 to a transport protein to form a S. aureus Type 5 capsular saccharide conjugate; b) conjugation of a S. aureus Type 8 capsular saccharide to a transport protein to form a S. aureus type 8 capsular saccharide conjugate, and c) combination of S. aureus Type 5 capsular saccharide conjugate, S. aureus Type 8 capsular saccharide conjugate, ClfA protein or fragment thereof and alpha toxoid to form the immunogenic composition.
Description of figures
Figure 1. Percentage of subjects experiencing pain after 1 or 2 doses of AC vaccine. In each formulation group, the first three columns provide the% of subjects experiencing pain after a single dose with the first column representing all pain reports, the second column representing pain greater than or equal to grade 2, and the third column representing grade 4 pain. The 4th, 5th and 6th columns show the same information after the second dose.
Figure 2. Percentage of subjects experiencing redness after 1 or 2 doses of 4C vaccine. In each formulation group, the first three columns provide the% of subjects experiencing redness after a single dose with the first column representing all redness ratios, the second column representing more than 50 mm of redness, and the third column representing more than 100 mm redness. The 4th, 5th and 6th columns show the same information after the second dose.
Figure 3. Percentage of subjects experiencing swelling after 1 or 2 doses of AC vaccine. In each formulation group, the first three columns provide the% of subjects experiencing swelling after a single dose with the first column representing all swelling ratios, the second column representing more than 50 mm swelling and the third column representing more than 100 mm swelling. The 4th, 5th and 6th columns show the same information after the second dose.
Figure 4. Immunogenicity results for S. aureus Type 5 capsular polysaccharide antibodies. GMC results from a Luminex test detecting antibodies against Type 5 capsular polysaccharide at various time points after the first and second immunizations are presented. The time points selected 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 chart) , day 14 after two immunizations (corresponding to day 44 on the chart), and day 30 after two immunizations (corresponding to day 60 on the chart). For each point of time, the results are presented in order (from left to right) for 5/10, 5 / 10AS, 10/30, 10 / 30AS and physiological saline.
Figure 5. Immunogenicity results for S. aureus Type 8 capsular polysaccharide antibodies. GMC results from a Luminex assay detecting antibodies against Type 8 capsular polysaccharide at various time points after the first one. and second immunizations are presented. The time points selected 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 chart) , day 14 after two immunizations (corresponding to day 44 on the chart), and day 30 after two immunizations (corresponding to day 60 on the chart). For each point of time, the results are presented in order (from left to right) for 5/10, 5 / 10AS, 10/30, 10 / 30AS and physiological saline.
Figure 6. Immunogenicity results for antibodies to S. aureus alpha toxoid. The GMC results of a Luminex test detecting antibodies against alpha toxoid at various points in time after the first and second immunizations are presented. The time points selected 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 chart) , day 14 after two immunizations (corresponding to day 44 on the chart), and day 30 after two immunizations (corresponding to day 60 on the chart). For each point of time, the results are presented in order (from left to right) for 5/10, 5 / 10AS, 10/30, 10 / 30AS and physiological saline.
Figure 7. Immunogenicity results for antibodies to S. aureus ClfA. The GMC results of an ELISA test detecting antibodies against ClfA at various points of time after the first and second immunizations are presented. The time points selected 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 chart) , day 14 after two immunizations (corresponding to day 44 on the chart), and day 30 after two immunizations (corresponding to day 60 on the chart). For each point of time, the results are presented in order (from left to right) for 5/10, 5 / 10AS, 10/30, 10 / 30AS and physiological saline.
Figure 8. Immunogenicity results for capsular polysaccharide of S. aureus Type 5 (panel A), capsular polysaccharide of S. aureus Type 8 (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
The present invention describes a method of immunizing against Staphylococcus aureus infection comprising a step of administering to a human patient a single dose of an immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a transport protein to form a capsular saccharide conjugate of S. aureus Type 5, wherein the capsular saccharide conjugate of S. aureus Type 5 is administered at a saccharide dose of 3 to 50 μg, 3 to 25 μg, 3 to 20 μg, 3 at 12 μg, 5 to 50 μg, 5 to 25 μg, 5 to 20 μg, 5 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
In one embodiment, the immunogenic composition further comprises a capsular saccharide of S. aureus Type 8 conjugated to a transport protein to form a capsular saccharide conjugate of S. aureus Type 8, wherein the capsular saccharide conjugate of S. aureus Type 8 is administered at a saccharide dose of 3 to 50 μg, 3 to 25 μg, 3 to 20 μg, 3 to 12 μg, 5 to 50 μg, 5 to 25 μg, 5 to 20 μg, 5 to 12 μg 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
In one embodiment, the same saccharide dose of capsular saccharide conjugate of S. aureus Type 5 and capsular saccharide conjugate of S. aureus Type 8 is present in the immunogenic composition; for example a dose of 4, 5, 6, 7, 8, 9 or 10 μg of saccharide of both Type 5 and Type 8 conjugates.
Most strains of S. aureus contain either 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 the structures of capsular polysaccharides as:
Type 5 -4) -β-D-ManNAcA- (1-4) -α-L-FucNAc (30Ac) - (1-3) -β-D-FucNAc- (1-Type 8 -3) -β- D-ManNAcA (40AC) - (1-3) -aL-FucNAc (1-3) -Ad-FucNAc (1-
The polysaccharides can be extracted from the appropriate strain of S. aureus using methods well known to those skilled in the art, for example as described in US 6,294,177, 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 they can be reduced in size, for example by microfluidization, ultrasonic irradiation or chemical treatment as exposure to a pH of 5.0 to 3.0. The invention also covers oligosaccharides derived from Type 5 and 8 polysaccharides derived from S. aureus. In one embodiment, the capsular saccharide of S. aureus Type 5 has a molecular weight greater than 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa or 90 kDa or between 25 and 125 kDa , 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa. In one embodiment, the capsular saccharide of S. aureus Type 8 has a molecular weight greater than 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa or 90 kDa or between 25 and 125 kDa. , 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
In one embodiment, the transport 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, CRM197, alpha toxoid, ClfA and Exoprotein A of Pseudomonas aeruginosa.
The type 5 and / or 8 capsular polysaccharide or the oligosaccharides included in the immunogenic composition of the invention are O-acetylated. In one embodiment, the degree of O-acetylation of the Type 5 capsular polysaccharide or oligosaccharide is 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%. , 50 to 90%, 60 to 90%, 70 to 90%, 70 to 80% or 80 to 90%. In one embodiment, the degree of O-acetylation of the Type 8 capsular polysaccharide or oligosaccharide is 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%. , 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 50 to 90%, 60 to 90%, 70 to 90%, 70 to 80% or 80 to 90%. In one embodiment, the degree of O-acetylation of Type 5 and Type 8 capsular polysaccharides or oligosaccharides is from 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, and 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 50 to 90%, 60 to 90%, 70 to 90%, 70 to 80% or 80 to 90%. In one embodiment, Type 5 and / or Type 8 capsular polysaccharides are 0 to acetylated at 80 to 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 Lemerciner 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.
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, 5048-5054) or treatment with 0.1N 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 which will lead to hydrolysis of 0-acetyl groups are minimized. For example, treatments with pH extremes are minimized.
Among the problems associated with the use of polysaccharides in vaccination is the fact that the polysaccharides per se are poor immunogens. Strategies, which have been designed to overcome this lack of immunogenicity, include binding the polysaccharide to large protein media, which provide help from nearby T cells. In one embodiment, the polysaccharides used in the invention are linked to a protein carrier, which provides help from nearby T cells. Examples of such carriers which can be used for coupling to polysaccharide or oligosaccharide immunogens include diphtheria and tetanus toxoids (DT, DT Crml97 and TT), keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa exoprotein A ( rEPA) and the purified protein derivative of tuberculin (PPD), Haemophilus influenzae protein D, pneumolysin or fragments of any of the above carriers. Fragments suitable for use include fragments encompassing helper T epitopes. In particular, a fragment of the protein D will possibly contain the N-terminal 1/3 of the protein. Protein D is an IgD binding protein of Haemophilus influenzae (EP 0 594 610 B1).
A novel transport protein that would be particularly advantageous for use in the context of antistaphylococcal 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 His35Leu or His35Arg variants is used as a carrier since the residual toxicity is lower. Alternatively, the alpha toxin is chemically detoxified by treatment with a crosslinking agent, 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 a crosslinking agent, formaldehyde or glutaraldehyde to further reduce toxicity.
The polysaccharides may be bound to one or more transport proteins by any known method (eg, Likhite, US Pat. No. 4,372,945 to Armor et al., US Patent 4,474,757, Anderson et al., WO 10/151544, Berti and 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 conjugation to CDAP, the cyanylation reagent 1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP) is optionally used for the synthesis of polysaccharide-protein conjugates. The cyanylation reaction can be carried out under relatively mild conditions, which avoids the hydrolysis of the alkali sensitive polysaccharides. This synthesis allows direct coupling to a transport protein.
The polysaccharide may be solubilized in water or saline. The CDAP can be dissolved in acetonitrile and added immediately to the solution of the polysaccharide. The CDAP reacts with the hydroxyl groups of the polysaccharide to form a cyanate ester. After the activation step, the transport protein is added. The amino groups of lysine react with the activated polysaccharide to form a covalent iso-urea bond. Following 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 permeation column to remove unreacted transport protein and residual reagents.
In one embodiment, the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus
Type 8 is directly conjugated to the transport protein. 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, the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus
Type 8 is conjugated using a cyanylation 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 the polysaccharide to the protein in the S. aureus Type 5 capsular saccharide conjugate is between 1/5 and 5/1 (w / w), 1/1 and 1/5 (p / p), 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 ( p / p). In one embodiment, the ratio of the polysaccharide to the protein in the capsular saccharide conjugate of S. aureus Type 8 is between 1/5 and 5/1 (ρ / ρ), 1/1 and 1/5 (p / p), 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 ( w / w).
Agglutination factor A (ClfA) has been identified as a S. aureus fibrinogen binding protein (US 6,008,341) and has been identified as a potential transport protein for polysaccharides that could be used to immunize against staphylococcal infection (WO 04/80490). ClfA is a surface-localized protein and is an important virulence factor because of its fibrinogen binding and S. aureus adhesion contribution. ClfA contains a fibrinogen binding region. This region, known as Domain A, is localized to the N-terminus of ClfA and comprises three separately folded subdomains, known as NI, N2, and N3. Domain A is followed by a serine-aspartate repetition region and a cell-wall and cell-membrane-containing region that contains the LPXTG motif for cell wall-mediated sorting. ClfA binds to the C-terminus of the γ chain of fibrinogen, and in this way is able to induce agglutination of bacteria in a fibrinogen solution (McDevitt et al (1997) Eur J. Biochem. 247, 416-424). Amino acid residues 221 to 559 of ClfA correspond to the N2-N3 region which is the smallest truncated product shown to retain fibrinogen binding. The fragments containing amino acids 221 to 559 of ClfA are the preferred fragments. Amino acid residues 532 to 538 correspond to the peptide peptide region of ClfA. Each subdomain comprises nine β-strands that form a new IgG-like folding. The peptide binding site of the γ chain of fibrinogen in ClfA is located in a hydrophobic groove at the junction between N2 and N3. Recently, ClfA amino acids P336 and Y338 have been recognized as fibrinogen binding sites, the mutation of which has led to fibrinogen binding loss (Josefsson et al., 2008, PLOS One Volume 3, Issue 5, page 1 -7). SEQ ID NOs: 8-12 contain point mutations at positions 336 and 338. The loss of fibrinogen binding in these variants led to an increase in the ability to protect against septic death in immunized mice, leading to concluding that the vaccine potential of recombinant ClfA is improved by eliminating its ability to bind fibrinogen (WO 09/95453). In one embodiment, the immunogenic composition further comprises a ClfA protein or a fragment thereof, 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 with the SEQ ID polypeptide sequence. NO: 3, 4, 5, 6 or 7 or 8 to 12 over their entire length. "Identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as may be the case, as measured by sequence comparison. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as may be the case, determined by the correspondence between the chains of such sequences. "Identity" can be easily 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: Informaties and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The methods for determining identity are designed to give the greatest
correspondence between the sequences tested. In addition, the methods for determining the identity are codified in computer programs available to the public. The methods of the computer programs for determining the identity between two sequences include, but are not limited to, the GAP program in the GCG package (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 family of BLAST programs 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 well-known Smith Waterman algorithm can also be used to determine identity.
The parameters for the comparison of polypeptide sequences include the following:
Algorithm: Needleman and Wunsch, Mol Mol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and
Henikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Breach Penalty: 8 Breach Length Penalty: 2
A useful program with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The above parameters are the default settings for peptide comparisons (without any penalty for tip gaps).
The parameters for the comparison of polynucleotides include the following:
Algorithm: Needleman and Wunsch, Mol Mol. 48: 443-453 (1970)
Comparison Matrix: Matches = +10, Mismatch = 0 Penalty Breach: 50 Penalty Breach Length: 3
Available as: gap program from
Genetics Computer Group, Madison WI. These are the default settings for nucleic acid comparisons.
Where a protein is specifically mentioned herein, it may be a reference to a native or recombinant full length protein or possibly to a mature protein in 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 of 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 from the d amino acids of the protein. In addition, such immunogenic fragments are generally immunologically reactive with antibodies generated against staphylococcal proteins or with antibodies generated by infection of a mammalian host with staphylococci or they contain T cell epitopes. In one embodiment, immunogenic fragments also include fragments which, when administered at an effective dose, (either alone or in the form of a hapten bound to a support), elicit a protective immune response against a staphylococcal infection, possibly they are protective against infection with S. aureus and / or S. epidermidis. Such an immunogenic fragment includes, for example, the protein lacking an N-terminal leader, and / or a transmembrane domain and / or a C-terminal anchor domain. For ClfA, the preferred fragments lack the repeat domain SD towards the C-terminal end of ClfA (for example using a fragment in which amino acids 555 to 927, 556 to 927, 557 to 927, 558 to 927 , 559 to 927 or 560 to 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 N-terminal methionine residue to allow recombinant expression.
In one embodiment, the immunogenic compositions of the invention may contain fusion proteins or ClfA fragments. The fusion protein optionally contains heterologous sequences as a provider of T-cell epitopes or purification markers, for example: β-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), epitope markers such as FLAG, myc tag, poly-histidine, or viral surface proteins such as hemagglutinin of influenza virus, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM197. The fusion protein is present in the immunogenic composition of the invention as a free protein or it may be a saccharide-related transport protein.
In one embodiment, the invention also provides an immunogenic fragment of the ClfA protein, i.e., a 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. That is, 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 includes, for example, the ClfA polypeptide lacking an N-terminal leader sequence, and / or the SD repeats 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. identity, most preferably 97 to 99% identity or 100% identity, with the amino acid sequence of any one of SEQ ID NO: 4 to 12 over the entire length of said sequence.
The fragments may be "independent" 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 in a larger single polypeptide. Other ClfA fragments comprise an isolated polypeptide comprising an amino acid sequence comprising 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 a ClfA fragment comprising the NI domain, the N2 domain, the N3 domain, the NI and N2 domains, the N2 and N3 domains or the Ni and N2 and N3 domains. Optionally, the ClfA fragment comprises the N2 and N3 domains and has an amino acid sequence at least identical to 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 6, 7, 11 or 12.
In one embodiment, the ClfA protein or 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 generally 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, Tyr338 amino acid of ClfA is mutated. In one embodiment, both Pro336 and Tyr338 are mutated, optionally to Alanine or Serine. In one embodiment, ClfA contains two mutations with mutated Pro336 in Ser and Tyr338 mutated to Ala.
In one embodiment, the ClfA protein or fragment is present in the immunogenic composition as an unconjugated protein. Alternatively, it is present conjugated to the capsular saccharide of S. aureus Type 5 or capsular saccharide of S. aureus Type 8. In such cases, ClfA can act as a transport protein and an antigen.
In one embodiment, the ClfA protein or fragment thereof is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg.
Alpha toxin is an important virulence determinant 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. In order to use alpha toxin in an immunogenic composition, it is generally 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 of at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 1 or 2.
The high toxicity of the 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 agents or by chemical conjugation to 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 be very important for the correct oligomerization required for pore formation and the mutation of this residue leads to the loss of toxicity. The modification of histidine 35 may be a substitution by Lys, Arg, Ala, Leu or Glu. A point mutation of the alpha toxin at Asp24, Lys37, His48, Lys58, Aspl00, Ilel07, Glul11, Metll3, Aspl27,
Aspl28, Gly130, Gly14, Hisl44, Lys147, Gln15, Aspl52, Phel53, Lys144, Vall69, Asn173, Arg200, Asn214, Leu219 or His259 may optionally be used to reduce toxicity.
When incorporated into immunogenic compositions of the 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 the S. aureus Type 5 polysaccharide and / or the S. aureus Type 8 polysaccharide.
In one embodiment, the immunogenic composition comprises alpha toxoid which contains a point mutation which decreases the toxicity of the alpha toxin, for example at the level of the amino acid 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 an unconjugated protein. Alternatively, the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8.
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 or 20 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 of the invention is mixed with a pharmaceutically acceptable excipient, and optionally with an adjuvant to form a vaccine.
The vaccines of the present invention may be combined with adjuvants, particularly when they are intended for use in populations of the elderly, immunocompromised individuals or those suffering from a chronic disease (such as diabetes, stage renal disease). terminal 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 a metabolizable oil (eg, squalene), an emulsifying agent (e.g., polyoxyethylene sorbitan monooleate) and optionally a tocol (e.g., alpha-tocopherol) are also suitable (WO 09/95453).
It is preferred that the adjuvant be chosen to be a preferential inducer of a response type TH1. Such 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 response is not absolute. In reality, an individual will support an immune response that is described as predominantly Th1 or predominantly Th2. However, it is often convenient to consider the cytokine families in terms of those described in murine CD4-positive T cell clones by Mosmann and Coffman (Mosmann, TR and Coffman, RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion
Immunology, 7, pl45-173). Traditionally, Th1-type responses are associated with the production of INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by lymphocytes. T, like the IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10. Suitable adjuvant systems that promote a predominantly Th1 response include: monophosphoryl lipid A or a derivative thereof (or detoxified lipid A in general - see, for example, WO 2005107798), particularly monophosphoryl lipid A 3-des-O-acylated (3D-MPL) (for preparation, see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with either an aluminum salt (for example, aluminum phosphate or aluminum hydroxide), or an oil emulsion in water. In such combinations, antigen and 3D-MPL are contained in the same particle structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further amplify the immunogenicity of an antigen adsorbed on alum [Thoelen et al. Vaccine (1998) 16: 708-14; EP 689454-B1].
An amplified 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. A particularly potent adjuvant formulation involving 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 CpGs (WO 96/02555) and other immunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are also preferred inducers of a TH1 response and are suitable for use in the present invention.
However, the inventors have discovered that in a clinical trial, the addition of an adjuvant as an oil-in-water emulsion did not produce an increase in immunogenicity. In view of the increase in reactogenicity that may be associated with the use of an adjuvant, an embodiment of the invention uses an immunogenic composition not supplemented with adjuvants, for example an immunogenic composition in which none of the components Staphylococci present is adsorbed on an adjuvant or an immunogenic composition in which the staphylococcal components are not mixed with an adjuvant in the form of an oil-in-water emulsion. The staphylococcal components comprise 1, 2, 3 or 4 of a S. aureus Type 5 capsular saccharide conjugate, a S. aureus type 8 capsular saccharide conjugate, a ClfA fragment or a of their fragments and an alpha toxoid.
Another aspect of the invention is a vaccine comprising the immunogenic composition described above and a pharmaceutically acceptable excipient. Vaccine preparations of the present invention can be used to protect or treat a human susceptible to S. aureus infection by administering said vaccine systemically or mucosally. Such administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or by mucosal administration to the oral / food, respiratory, genitourinary systems.
Vaccine preparation is generally described in Vaccine Design ("The Subunit and Adjuvant Approach" (Powell eds M.F. & Newman M.J.) (1995) Plenum Press New York). The encapsulation within liposomes is described by Fullerton, U.S. Patent 4,235,877.
The vaccines of the present invention can 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 it is lyophilized and reconstituted extemporaneously before use. Lyophilization may result in a more stable composition (vaccine). The invention also encompasses a method of making the immunogenic compositions and vaccines of the invention. In one embodiment, the method of the invention is a method for making a vaccine comprising the steps of: a) conjugating a S. aureus Type 5 capsular saccharide to a transport protein to form a capsular saccharide conjugate S. aureus Type 5, b) conjugation of a capsular saccharide of S. aureus Type 8 to a transport protein to form a capsular saccharide conjugate of S. aureus Type 8, and c) combination of the capsular saccharide conjugate of S. aureus Type 5, capsular saccharide conjugate of S. aureus Type 8, a ClfA protein or 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 infection, particularly nosocomial infections acquired in hospital.
The immunogenic composition or vaccine of the invention is particularly advantageous for use in non-emergency surgery cases, particularly when subjects are immunized with a single dose. Such patients will know the date of the surgery in advance and can be inoculated advantageously in advance. In one embodiment, the subject is immunized with a single dose of the immunogenic composition of the invention at 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 invention at 60, 6 to 40, 7 to 30 or 7 to 15 days prior to a planned procedure at the hospital, for example a surgical procedure as a cardio-thoracic surgical procedure. Generally, adults over the age of 16 waiting for elective surgery are treated with the immunogenic compositions and vaccines of the invention. Alternatively, children aged 3 to 16 years waiting for elective surgery are treated with the immunogenic compositions and vaccines of the invention.
It is also possible to inoculate health professionals with the vaccine of the invention.
The vaccine preparations of the present invention can be used to protect or treat a human being susceptible to S. aureus infection by administering said vaccine systemically or mucosally.
Such administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or by mucosal administration to the oral / food, respiratory, genitourinary systems.
One embodiment of the invention is a method of preventing or treating an infection or a staphylococcal disease comprising administering the immunogenic composition or vaccine of the invention to a patient in need thereof.
Another embodiment of the invention is a use of the immunogenic composition of the invention in the manufacture of a vaccine for the treatment or prevention of an infection or a staphylococcal disease, possibly an infection. postoperative staphylococcal.
The terms "comprising", "include" and "includes" herein are intended by the inventors as possibly substitutable by the terms "consisting of", "consist of" and "consists of", respectively, in all cases . However, the terms "including", "understand" and "understand" retain their usual "open" meaning when they have not been substituted.
All references or patent applications cited in this patent specification are incorporated herein by reference.
For this invention to be better understood, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.
Examples
Example 1 Sequences of the Proteins SEQ ID NO: 1
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSF
IDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTK
EYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQN
WGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNID
VIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 2 - Alpha toxin
MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV
YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGL
IGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGS
MKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTK
DKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 3 - ClfA of S. aureus strain NCTC8325
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKT DDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNT NAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDWNQ AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKG DTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYID PENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVI APVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEF NTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVV PEQPDEPGEIEPIPEDSDSDPGSDSGSDSNSDSGSDSGSDSTSDSGSDSASDSDSASDSDSASDSDSA S DS DSAW DS DS DNDS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS PS DS DS DS DS DS DS DS DS DS DS PS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS DS SDSDSDSDSDSDSDSASDSDSGSDSDSSSDSDSESDSNSDSESVSNNNVVPPNSPKNGTNASNKNEAK DSKEPL PDTGSEDEANTSLIWGLLASIGSLLLFRRKKENKDKK
SEQ ID NO: 4 - N1N2N3 from ClfA
MSENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNA TTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVST TQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVG IDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVID SDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNL SIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSE SYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE
SEQ ID NO: 5 - Nl-3 from ClfA
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKT DDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNT NAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQ AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKG DTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYID PENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVI APVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEF NTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVV PEQPDEPGEIEPIPE
SEQ ID NO: 6 - N23 from ClfA
SLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELN LNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTL ATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNT DSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYI VVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEP IPE
SEQ ID NO: 7 - shorter N23 from ClfA
GTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPP IMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKT VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTS IKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSK GDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 8 - ClfA strain of S. aureus NCTC8325
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKT DDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNT NAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDWNQ AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKG DTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAID PENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVI APVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEF NTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVV PEQPDEPGEIEPIPEDSDSDPGSDSGSDSNSDSGSDSGSDSTSDSGSDSASDSDSASDSDSASDSDSA SDSDSASDSDSDNDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDSASDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSESDSDSDSD SDSDSDSDSDSDSDSASDSDSGSDSDSSSDSDSESDSNSDSESVSNNNVVPPNSPKNGTNASNKNEAK DSKEPLPDTGSEDEANTSLIWGLLASIGSLLLFRRKKENKDKK
SEQ ID NO: 9 - N1N2N3 from ClfA
MSENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNA TTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVST TQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVG IDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVID SDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNL
SIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSE SYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE
SEQ ID NO: 10 - Nl-3 from ClfA
MNMKKKEKHAIRKKSIGVASVLVGTLIGFGLLSSKEADASENSVTQSDSASNESKSNDSSSVSAAPKT DDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNT NAEELVNQTSNETTSNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQ AVNTSAPRMRAFSLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKG DTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAID PENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVI APVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEF NTPDDQITTPYIVWNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVV PEQPDEPGEIEPIPE
SEQ ID NO: 11 - N23 from ClfA
SLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELN LNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTL ATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNT DSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYI VVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEP IPE
SEQ ID NO: 12 - N23 shorter ClfA
GTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPP
IMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKT
VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTS
IKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSK
GDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 13
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMRK KVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQI SDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESP TDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSS GFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKI DWEKEEMTN SEQ ID NO: 14
MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMRKKVFYSFIDDKNHNKKLLVIRTKGTI AGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGN VTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRD SWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDV IYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 15
MASLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVT STAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKT VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVD NAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 16
MAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMA
GDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGK
FYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYF
VNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWD
NEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 17
MASLAAVAADAPVAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVT STAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKT VLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVD NAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNS NIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE SEQ ID NO: 18
MAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGD
QVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMSAAIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFY
NLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVN
PENFEDVTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNE
VAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPE
Example 2 - Preparation of the vaccine components
A four-component antistaphylococcal vaccine was prepared which contained a capsular polysaccharide of S. aureus Type 5 conjugated to a transport protein, tetanus toxoid, capsular polysaccharide of S. aureus Type 8 conjugated to a transport protein, and tetanus toxoid, a ClfA fragment containing the N2 and N3 domains and point mutations at residues 336 and 338 where 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 changed to arginine. Capsular polysaccharides were conjugated to tetanus toxoid using CDAP as the coupling agent. This process is described in WO 07/113222. Alpha toxoid and ClfA proteins can be made using conventional systems of molecular biology and expression.
Four formulations of the antistaphylococcal vaccine were manufactured: 5/10 contained: 5 μg of conjugated saccharide dose
Type 5 - 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 product described above. 10/30 contained: 10 μg of Type 5 tetanus toxoid saccharide dose, 10 μg of Type 8 tetanus toxoid saccharide dose, 30 μg of alpha toxoid and 30 μg of the cleaved product of ClfA described below. above. 5 / 10AS contained: 5 μg of Type 5 tetanus toxoid saccharide dose, 5 μg of Type 8 tetanus toxoid saccharide dose, 10 μg of alpha toxoid and 10 μg of the truncated ClfA product described below. and as an adjunct an oil-in-water emulsion containing squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate. 10 / 30AS contained: 10 μg of Type 5 tetanus toxoid saccharide dose, 10 μg of Type 8 tetanus toxoid saccharide dose, 30 μg of alpha toxoid and 30 μg of the truncated ClfA product described below. and as an adjunct an oil-in-water emulsion containing squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate.
Example 3 - Results of a clinical trial using the 4 component antistaphylococcal vaccine
A Phase I clinical trial was conducted using a total of 88 healthy adults aged 18 to 40 years. The control group contained 30 subjects who were inoculated with saline. The remaining subjects were divided into four arms with 15/14 subjects immunized with each of the formulations described in Example 2 (5/10, 5 / 10AS, 10/30 and 10 / 30AS). Vaccine doses were given at the start of the trial and after one month and at six months. Blood samples for the humoral analysis were taken on days 0, 7, 14 and 30 after each dose and on days 360 and 540.
The details of the topics are provided below.
Group N Average age% of women 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 Serum 30 30.1 50 physiological Reactogenicity and safety
The 4-component antistaphylococcal vaccine was generally safe and well tolerated. After the first and second doses, no serious adverse event and no potential immune mediated disorder were observed. The percentage of subjects reporting pain, redness and swelling after dose 1 and dose 2 is shown in Figures 1 to 3. Pain was experienced at the injection site in 78.6 to 100% of subjects in the vaccine group compared to 3 to 4% in the control group (see Figure 1). However, only one case was graded 3. The results for the incidence of redness and swelling are shown in Figures 2 and 3. For both parameters, there was a higher incidence trend of redness / swelling after the second dose compared with a single dose for the 10/30 arm of the study.
immunogenicity
Blood samples taken at day 0 and 7, 14 and 30 days after the second and third immunizations were tested by Luminex or ELISA to establish the level of IgG produced against each antigen of the four-component antistaphylococcal vaccine.
The results for immunogenicity are shown in Figures 4 to 8 and Tables 1 to 5 below.
Prevalence, there was 83.3 to 100% seropositivity for all tests. Despite considerable levels of background immunity, the 4-component vaccine was able to elicit a strong immune response against the 4 components.
Figures 4 to 7 show that for CPS5, CPS8, alpha toxoid and ClfA, the first immunization produced the largest increase in immunogenicity with large increases in obvious GMCs at days 14 and 30. The second immunization at day 30 did not produce any 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 did not cause further increase in GMCs with GMC levels remaining approximately the same for all four components between day 30 and day 540. Therefore, a single immunization is an effective means of producing a maximal immune response.
Immunogenicity results for the 10/30 assay appear to be stronger than for the 5/10 assay with an approximately 1.5 to 2 fold increase in GMC for CPS5, CPS8 and alpha toxoid. In the case of ClfA, the increase in GMC was approximately 3.8 times at the higher dose. The addition of an oil-in-water emulsion adjunct did not increase the immunogenicity of the 4-component vaccine, as demonstrated by a comparison of the 5/10 antibody-mediated antibody response. and 5 / 10AS and the 10/30 and 10 / 30AS arms.
Table 1
Seropositivity and GMC for IgG Ac antibodies against Staphylococcus aureus CPS 5 (protocol compliant cohort for immunogenicity)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of anatoxin a
5 / 10AS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid a, adjuvanted with AS03B 10/30 = 10 μg of CPS5-TT, 10 μg of CPS8-TT , 30 g of ClfA, 30 g of toxoid at 10/30 AS = 10 g of CPS5-TT, 10 g of CPS8-TT, 30 g of ClfA, 30 g of toxoid a, adjuvanted
by AS03B Physiological serum = pool of saline 1 and saline 2 GMC = geometric mean of antibody concentration calculated on all subjects N = number of subjects with available results n /% = number / percentage of subjects with concentration within the specified 95% CI range = 95% confidence interval; LL = lower limit, UL = upper limit PRE = pre-dose 1 PI (J7) = 7 days post-dose 1 PI (J14) = 14 days post-dose 1 PI (J30) = 30 days post-dose 1 (sample blood taken at visits 5 or 6) PII (J37) = 7 days post-dose 2 PII (D44) = 14 days post-dose 2 PII (D60) = 30 days post-dose 2
Table 2
Seropositivity and GMC for IgG Ac antibodies to Staphylococcus aureus CPS 8 (protocol compliant cohort for immunogenicity)
5/10 = 5 μl of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid a
5 / 10AS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid a, adjuvanted with λS03B 10/30 = 10 μg of CPS5-TT, 10 μg of CPS8-TT , 30 μg of ClfA, 30 μg of 10 / 30AS toxoid = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid a, adjuvanted
by AS03B Physiological serum = pool of saline 1 and saline 2 GMC = geometric mean of antibody concentration calculated on all subjects N = number of subjects with available results n /% = number / percentage of subjects with concentration within the specified 95% CI range = 95% confidence interval; LL = lower limit, UL = upper limit PRE = pre-dose 1 PI (J7) = 7 days post-dose 1 PI (J14) = 14 days post-dose 1 PI (J30) = 30 days post-dose 1 (sample blood taken at visits 5 or 6) PII (J37) = 7 days post-dose 2 PII (D44) = 14 days post-dose 2 PII (D60) = 30 days post-dose 2
Table 3
Seropositivity and GMC for IgG Ab antibodies to Staphylococcus aureus alpha toxin (protocol compliant cohort for immunogenicity)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of anatoxin a
5 / 10AS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid α, adjuvanted with AS03B 10/30 = 10 μg of CPS5-TT, 10 μg of CPS8-TT , 30 μg of ClfA, 30 μg of 10 / 30AS toxoid = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid a, adjuvanted
by AS03B Physiological serum = pool of saline 1 and saline 2 GMC = geometric mean of antibody concentration calculated on all subjects N = number of subjects with available results n /% = number / percentage of subjects with concentration within the specified 95% CI range = 95% confidence interval; LL = lower limit, UL = upper limit PRE = pre-dose 1 PI (J7) = 7 days post-dose 1 PI (J14) = 14 days post-dose 1 PI (J30) = 30 days post-dose 1 (sample blood taken at visits 5 or 6) PII (J37) = 7 days post-dose 2 PII (D44) = 14 days post-dose 2 PII (D60) = 30 days post-dose 2
Table 4
Seropositivity and GMC for IgG Ac antibodies against Staphylococcus aureus ClfA (protocol compliant cohort for immunogenicity)
5/10 = 5 μl of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of 5 / 10AS toxoid = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of CPS5-TT, ClfA, 10 μg of toxoid a, adjuvanted by
ÄS03B 10/30 = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of 10 / 30AS toxoid = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid a, adjuvanted
by AS03B Physiological serum = pool of saline 1 and saline 2 GMC = geometric mean of antibody concentration calculated on all subjects N = number of subjects with available results n /% = number / percentage of subjects with concentration within the specified 95% CI range = 95% confidence interval; LL = lower limit, UL = upper limit PRE = pre-dose 1 PI (J7) = 7 days post-dose 1 PI (J14) = 14 days post-dose 1 PI (J30) = 30 days post-dose 1 (sample of blood taken at visits 5 or 6) PII (J37) = 7 days post-dose 2 PII (J44i = 14 days post-dose 2 PII (J60) = 30 days post-dose 2
Table 5
Seropositivity and GMC for IgG Ac antibodies against C. tetani toxin (protocol compliant cohort for immunogenicity)
5/10 = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of anatoxin a
5 / 10AS = 5 μg of CPS5-TT, 5 μg of CPS8-TT, 10 μg of ClfA, 10 μg of toxoid α, adjuvanted with AS03B 10/30 = 10 μg of CPS5-TT, 10 μg of CPS8-TT , 30 μg of ClfA, 30 μg of 10 / 30AS toxoid = 10 μg of CPS5-TT, 10 μg of CPS8-TT, 30 μg of ClfA, 30 μg of toxoid a, adjuvanted
by AS03B Physiological serum = pool of saline 1 and saline 2 GMC = geometric mean of antibody concentration calculated on all subjects N = number of subjects with available results n /% = number / percentage of subjects with concentration within the specified 95% CI range = 95% confidence interval; LL = lower limit, UL = upper limit PRE = pre-dose 1 PI (J7) = 7 days post-dose 1 PI (J14) = 14 days post-dose 1 PI (J30) = 30 days post-dose 1 (sample blood taken at visits 5 or 6) PII (J37) = 7 days post-dose 2 PII (D44) = 14 days post-dose 2 PII (D60) = 30 days post-dose 2

权利要求:
Claims (131)
[1]
A method of immunizing against Staphylococcus aureus infection comprising a step of administering to a human patient a single dose of an immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a transport protein to form a S. aureus type 5 capsular saccharide conjugate, where the capsular saccharide conjugate of S. aureus Type 5 is administered at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
[2]
The method of claim 1, wherein the immunogenic composition comprises a capsular saccharide of Staphylococcus aureus Type 8 conjugated to a transport protein to form a capsular saccharide conjugate of S. aureus Type 8, wherein the S capsular saccharide conjugate Aureus Type 8 is administered at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg. pg.
[3]
The method of claim 1 or 2, wherein the capsular saccharide of S. aureus Type 5 has a molecular weight greater than 25 kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[4]
The method according to any one of claims 1 to 3, wherein the capsular saccharide of S. aureus Type 8 has a molecular weight greater than 25 kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[5]
The method according to any one of claims 1 to 4, wherein the transport protein to which the Type 5 capsular saccharide is conjugated is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
[6]
The method according to any one of claims 1 to 5, wherein the transport protein to which the Type 8 capsular saccharide is conjugated is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
The method of claim 5 or 6, wherein the transport protein is tetanus toxoid.
[8]
The method according to any one of claims 1 to 7, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is 50-100% O-acetylated or 75-100%. %.
[9]
The method of any one of claims 1 to 8, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is conjugated directly to the transport protein.
[10]
The method of any one of claims 1 to 9, wherein the capsular saccharide of S. aureus Type 5 is conjugated using a cyanylation reagent.
[11]
The method of any one of claims 1 to 10, wherein the capsular saccharide of S. aureus Type 8 is conjugated using a cyanylation reagent.
[12]
The method of claim 10 or 11, wherein the cyanylation reagent is CDAP.
[13]
The method according to any one of claims 1 to 12, wherein the ratio of polysaccharide to protein in S. aureus Type 5 capsular saccharide conjugate is between 1/5 and 5/1 (w / w). or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w).
[14]
The method according to any one of claims 1 to 13, wherein the ratio of polysaccharide to protein in S. aureus Type 8 capsular saccharide conjugate is between 1/5 and 5/1 (w / w) or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w).
[15]
The method of any one of claims 1 to 14, wherein the same saccharide dose of capsular saccharide of S. aureus Type 5 and capsular saccharide of S. aureus Type 8 is present in the immunogenic composition.
[16]
The method of any one of claims 1 to 15, wherein the immunogenic composition further comprises a ClfA protein or fragment thereof.
[17]
The method of claim 16, wherein the ClfA protein or fragment thereof is at least 90% identical with the polypeptide sequence of any of SEQ ID NO: 3 to 12 over their entire length.
[18]
The method of claim 16 or 17, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the N2 domain.
[19]
19. The method according to claims 16 to 18, wherein the ClfA protein or one of its fragments is a ClfA fragment comprising the N3 domain.
[20]
The method of any one of claims 16 to 19, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the NI domain.
[21]
21. The method according to claim 16, wherein the ClfA protein or one of its fragments comprises the N2-N3 domains and has a polypeptide sequence at least 90% identical with the sequence of SEQ ID NO. : 6, 7, 11, 12, 15, 16, 17 or 18.
[22]
The method of any one of claims 16 to 21, wherein the ClfA protein or fragment thereof contains an amino acid substitution that reduces the ability of ClfA to bind to fibrinogen.
[23]
The method of claim 22, wherein the ClfA protein or fragment thereof contains an amino acid substitution at least one of the amino acids Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389. , Tyr474, Glu526 or Val527.
[24]
24. The method of claim 22, wherein the amino acid Pro336 is mutated to Ser and / or Y338 is mutated to Ala.
[25]
25. The method of any one of claims 16 to 24, wherein the ClfA protein or fragment thereof is present in the immunogenic composition as an unconjugated protein.
[26]
The method of any one of claims 16 to 24, wherein the ClfA protein or fragment thereof is conjugated to the capsular saccharide of S. aureus Type 5.
[27]
The method of any of claims 16 to 24, wherein the ClfA protein or fragment thereof is conjugated to the capsular saccharide of S. aureus Type 8.
[28]
28. A method according to any one of claims 16 to 18, wherein the ClfA protein or fragment thereof is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 pg.
[29]
29. A method according to any one of claims 1 to 28, wherein the single dose of the immunogenic composition is administered at 60, 6 to 40, 7 to 30 or 7 to 15 days before a planned procedure at the hospital.
[30]
30. A method according to any one of the preceding claims, wherein the immunogenic composition comprises an alpha toxoid.
[31]
31. The method of claim 30, wherein the alpha toxoid has an amino acid sequence at least 90% identical with SEQ ID NO: 1, 2, 13 or 14.
[32]
The method of claim 30 or 31, wherein the alpha toxoid contains a point mutation that decreases the toxicity of the alpha toxoid.
[33]
The method of claim 32, wherein the alpha toxoid contains a point mutation at amino acid level 35.
[34]
34. The method of claim 33, wherein the point mutation at the amino acid level replaces a histidine with an amino acid arginine.
[35]
35. A method according to any one of claims 30 to 34, wherein the alpha toxoid is present in the immunogenic composition as a non-conjugated protein.
[36]
36. The method according to any one of claims 30 to 35, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 5.
[37]
37. The method of any one of claims 30 to 36, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 8.
[38]
38. The method according to any one of claims 30 to 37, wherein the alpha toxoid is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg.
[39]
39. The method of any one of claims 30 to 38, wherein ClfA and alpha toxoid are present at the same dose in the immunogenic composition.
[40]
40. A process according to any one of the preceding claims, wherein the immunogenic composition does not contain oil-in-water emulsion.
[41]
41. The method of any one of the preceding claims, wherein the immunogenic composition is non-adjuvanted.
[42]
42. An immunogenic composition comprising a capsular saccharide of Staphylococcus aureus Type 5 conjugated to a transport protein to form a S. aureus Type 5 capsular saccharide conjugate, wherein the capsular saccharide conjugate of S. aureus Type 5 is administered at a dose of saccharide of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg, for use in the treatment or prevention of infection with Staphylococcus aureus, where a single dose of the immunogenic composition is administered to a human patient.
[43]
43. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 42, wherein the immunogenic composition comprises a capsular saccharide of S. aureus Type 8 conjugated to a transport protein to form a conjugate. capsular saccharide of S. aureus Type 8, wherein the capsular saccharide conjugate of S. aureus Type 8 is administered at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg, at 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
[44]
An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 42 or 43, wherein the capsular saccharide of S. aureus Type 5 has a molecular weight greater than 25 kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[45]
45. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 44, wherein the capsular saccharide of S. aureus Type 8 has a molecular weight greater than 25. kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[46]
An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 45, wherein the transport protein to which the Type 5 capsular saccharide is conjugated is selected in the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
[47]
An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 46, wherein the transport protein to which the Type 8 capsular saccharide is conjugated is selected in the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
[48]
48. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 46 or 47, wherein the transport protein is tetanus toxoid.
[49]
49. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 48, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is O-acetylated at 50 to 100% or 75 to 100%.
[50]
50. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 49, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is conjugated directly to the transport protein.
[51]
51. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 50, wherein the capsular saccharide of S. aureus Type 5 is conjugated using a reagent of cyanylating.
[52]
52. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 51, wherein the capsular saccharide of S. aureus Type 8 is conjugated using a reagent of cyanylating.
[53]
53. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 51 or 52, wherein the cyanylation reagent is CDAP.
[54]
54. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 53, wherein the ratio of polysaccharide to protein in S. capsular saccharide conjugate. Aureus Type 5 is between 1/5 and 5/1 (w / w) or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w).
[55]
55. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 54, wherein the ratio of polysaccharide to protein in S. capsular saccharide conjugate. Aureus Type 8 is between 1/5 and 5/1 (w / w) or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w).
[56]
56. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any of claims 42 to 55, wherein the same saccharide dose of the capsular saccharide of S. aureus Type 5 and the Capsular saccharide of S. aureus Type 8 is present in the immunogenic composition.
[57]
57. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 56, the immunogenic composition further comprising a ClfA protein or fragment thereof.
[58]
58. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 57, wherein the ClfA protein or fragment thereof is at least 90% identical with the polypeptide sequence of any of SEQ ID NO: 3 to 12 or 15 to 18 over their entire length.
[59]
59. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 57 or 58, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the N2 domain.
[60]
60. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 59, wherein the ClfA protein or fragment thereof is a fragment of ClfA comprising the N3 domain.
[61]
61. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 60, wherein the ClfA protein or fragment thereof is a fragment of ClfA comprising the Ni domain.
[62]
62. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any of claims 57 and 58, wherein the ClfA protein or fragment thereof comprises the N2-N3 domain. and has a polypeptide sequence at least 90% identical with the sequence of SEQ ID NO: 6, 7, 11, 12, 15, 16, 17 or 18.
[63]
63. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 62, wherein the ClfA protein or fragment thereof contains an acid substitution. amine which reduces the ability of ClfA to bind to fibrinogen.
[64]
64. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 63, wherein the ClfA protein or fragment thereof contains an amino acid substitution at minus one of the amino acids Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389, Tyr474, Glu526 or Val527.
[65]
65. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 63, wherein the amino acid Pro336 is mutated to Ser and / or Y338 is mutated to Ala.
[66]
66. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 65, wherein the ClfA protein or fragment thereof is present in the immunogenic composition in the form of an unconjugated protein.
[67]
An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 65, wherein the ClfA protein or fragment thereof is conjugated to the capsular saccharide of S. aureus Type 5.
[68]
68. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 65, wherein the ClfA protein or fragment thereof is conjugated to the capsular saccharide of S. aureus Type 8.
[69]
69. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 57 to 68, wherein the ClfA protein or a fragment thereof is present in the immunogenic composition. at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg.
[70]
70. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 69, wherein the single dose of the immunogenic composition is administered at 60, 6 to 40 hours. , 7 to 30 or 7 to 15 days before a planned procedure at the hospital.
[71]
71. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of the preceding claims, wherein the immunogenic composition comprises an alpha toxoid.
[72]
An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 71, wherein the alpha toxoid has an amino acid sequence of at least 90% with SEQ ID NO: 1, 2, 13 or 14.
[73]
73. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 71 or 72, wherein the alpha toxoid contains a point mutation which decreases the toxicity of the alpha toxoid.
[74]
74. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 73, wherein the alpha toxoid contains a point mutation at amino acid level 35.
[75]
75. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to claim 74, wherein the point mutation at the amino acid level replaces a histidine with an amino acid arginine.
[76]
76. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 71 to 75, wherein the alpha toxoid is present in the immunogenic composition in the form of a unconjugated protein.
[77]
77. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 71 to 76, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 5.
[78]
78. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 71 to 77, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 8.
[79]
79. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 71 to 78, wherein the alpha toxoid is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg.
[80]
80. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 71 to 79, wherein ClfA and alpha toxoid are present at the same dose in the composition. immunogenic.
[81]
81. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 80, wherein the immunogenic composition does not contain an oil-in-water emulsion.
[82]
82. An immunogenic composition for use in the treatment or prevention of S. aureus infection according to any one of claims 42 to 81, wherein the immunogenic composition is not supplemented with adjuvants.
[83]
83. An immunogenic composition comprising a Type 5 capsular saccharide conjugated to a transport protein, a Type 8 capsular saccharide conjugated to a transport protein, a ClfA protein or a fragment thereof and an alpha toxoid.
[84]
84. An immunogenic composition according to claim 83, wherein the capsular saccharide conjugate of S. aureus Type 5 is present at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 μg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
[85]
85. An immunogenic composition according to claim 83 or 84, wherein the capsular saccharide conjugate of S. aureus Type 8 is present at a saccharide dose of 3 to 50 μg, 5 to 25 μg, 3 to 20 μg, 3 to 12 pg, 5 to 10 μg, 7 to 20 μg, 7 to 15 μg or 8 to 12 μg.
[86]
86. An immunogenic composition according to any one of claims 83 to 85, wherein the capsular saccharide of S. aureus Type 5 has a molecular weight greater than 25 kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[87]
87. An immunogenic composition according to any one of claims 83 to 86, wherein the capsular saccharide of S. aureus Type 8 has a molecular weight greater than 25 kDa, 40 kDa or 50 kDa, or between 25 and 125 kDa, 90 and 125 kDa, 30 and 100 kDa, 35 and 75 kDa or 40 and 70 kDa.
[88]
88. An immunogenic composition according to any one of claims 83 to 87, wherein the transport protein to which the Type 5 capsular saccharide is conjugated is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
[89]
89. An immunogenic composition according to any one of claims 83 to 88, wherein the transport protein to which the Type 8 capsular saccharide is conjugated is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, alpha toxoid, ClfA, exoprotein A of Pseudomonas aeruginosa.
[90]
90. An immunogenic composition according to claim 88 or 89, wherein the transport protein is tetanus toxoid.
[91]
91. An immunogenic composition according to any one of claims 83 to 90, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is O-acetylated at 50 to 100% or 75 to 100%. 100%.
[92]
92. An immunogenic composition according to any one of claims 83 to 91, wherein the capsular saccharide of S. aureus Type 5 and / or the capsular saccharide of S. aureus Type 8 is conjugated directly to the transport protein.
[93]
93. An immunogenic composition according to any one of claims 83 to 92, wherein the capsular saccharide of S. aureus Type 5 is conjugated using a cyanylation reagent.
[94]
94. An immunogenic composition according to any of claims 83 to 93, wherein the capsular saccharide of S. aureus Type 8 is conjugated using a cyanylation reagent.
[95]
95. An immunogenic composition according to claim 93 or 94, wherein the cyanylation reagent is CDAP.
[96]
96. An immunogenic composition according to any of claims 83 to 95, wherein the ratio of polysaccharide to protein in S. aureus Type 5 capsular saccharide conjugate is between 1/5 and 5/1 (w / w). ) or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w) ·
[97]
97. An immunogenic composition according to any one of claims 83 to 96, wherein the ratio of polysaccharide to protein in S. aureus Type 8 capsular saccharide conjugate is between 1/5 and 5/1 (w / w). ) or between 1/2 and 2/1 (w / w), 1/2 to 1/5 (w / w) or 1/1 and 1/2 (w / w) -
[98]
98. An immunogenic composition according to any one of claims 83 to 97, wherein the same saccharide dose of capsular saccharide of S. aureus Type 5 and capsular saccharide of S. aureus Type 8 is present in the immunogenic composition.
[99]
99. Immunogenic composition according to any one of claims 83 to 98, the immunogenic composition further comprising a ClfA protein or a fragment thereof.
[100]
Immunogenic composition according to claim 99, wherein the ClfA protein or fragment thereof is at least 90% identical with the polypeptide sequence of any of SEQ ID NO: 3 to 12 or 15 to 18 on their entire length.
[101]
101. An immunogenic composition according to claim 99 or 100, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the N2 domain.
[102]
102. An immunogenic composition according to any one of claims 99 to 101, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the N3 domain.
[103]
103. An immunogenic composition according to any one of claims 99 to 102, wherein the ClfA protein or fragment thereof is a ClfA fragment comprising the NI domain.
[104]
104. An immunogenic composition according to any one of claims 99 to 103, wherein the ClfA protein or a fragment thereof comprises the N2-N3 domain and has a polypeptide sequence at least 90% identical with the sequence of SEQ ID NO: 6, 7, 11, 12, 15, 16, 17 or 18.
[105]
105. An immunogenic composition according to any one of claims 99 to 104, wherein the ClfA protein or fragment thereof contains an amino acid substitution which reduces the ability of ClfA to bind to fibrinogen.
[106]
An immunogenic composition according to claim 105, wherein the ClfA protein or fragment thereof contains an amino acid substitution at least one of the amino acids Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389, Tyr474, Glu526 or Val527.
[107]
107. An immunogenic composition according to claim 105, wherein the amino acid Pro336 is mutated to Ser and / or Tyr338 is mutated to Ala.
[108]
108. An immunogenic composition according to any one of claims 99 to 107, wherein the ClfA protein or fragment thereof is present in the immunogenic composition as an unconjugated protein.
[109]
109. The immunogenic composition according to any one of claims 99 to 107, wherein the ClfA protein or a fragment thereof is conjugated to the capsular saccharide of S. aureus Type 5.
[110]
110. An immunogenic composition according to any one of claims 99 to 107, wherein the ClfA protein or fragment thereof is conjugated to the capsular saccharide of S. aureus Type 8.
[111]
111. The immunogenic composition according to any one of claims 99 to 110, wherein the ClfA protein or a fragment thereof is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 at 40 pg.
[112]
112. Immunogenic composition according to claim 111, the immunogenic composition having a volume of 0.5 ml.
[113]
113. Immunogenic composition according to any one of claims 83 to 112, the immunogenic composition comprising an alpha toxoid.
[114]
The immunogenic composition of claim 113, wherein the alpha toxoid has an amino acid sequence of at least 90% identical with SEQ ID NO: 1, 2, 13 or 14.
[115]
115. An immunogenic composition according to claim 113 or 114, wherein the alpha toxoid contains a point mutation which decreases the toxicity of the alpha toxoid.
[116]
116. An immunogenic composition according to claim 115, wherein the alpha toxoid contains a point mutation at amino acid level 35.
[117]
117. An immunogenic composition according to claim 116, wherein the point mutation at the amino acid level replaces a histidine with an amino acid arginine or leucine.
[118]
118. An immunogenic composition according to any one of claims 113 to 117, wherein the alpha toxoid is present in the immunogenic composition as an unconjugated protein.
[119]
11. An immunogenic composition according to any one of claims 113 to 118, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 5.
[120]
120. An immunogenic composition according to any one of claims 113 to 119, wherein the alpha toxoid is conjugated to the capsular saccharide of S. aureus Type 8.
[121]
The immunogenic composition according to any one of claims 113 to 120, wherein the alpha toxoid is present in the immunogenic composition at a dose of 5 to 50, 10 to 30, 5 to 15 or 20 to 40 μg.
[122]
122. An immunogenic composition according to any one of claims 113 to 121, wherein ClfA and alpha toxoid are present at the same dose in the immunogenic composition.
[123]
123. Immunogenic composition according to any one of claims 83 to 122, wherein the immunogenic composition does not contain oil-in-water emulsion.
[124]
The immunogenic composition of any one of claims 83 to 123, wherein the immunogenic composition is not supplemented with adjuvants.
[125]
125. A vaccine comprising the immunogenic composition of any one of claims 83 to 124, and a pharmaceutically acceptable excipient.
[126]
A method of making the immunogenic composition according to any one of claims 83 to 124 or the vaccine of claim 125 comprising the steps of: a) conjugating a capsular saccharide of S. aureus Type 5 to a transport protein to form a S. aureus Type 5 capsular saccharide conjugate; b) conjugation of a S. aureus Type 8 capsular saccharide to a transport protein to form a S. aureus type 8 capsular saccharide conjugate, and c) combination of S. aureus Type 5 capsular saccharide conjugate, S. aureus Type 8 capsular saccharide conjugate, ClfA protein or fragment thereof and alpha toxoid to form the immunogenic composition.
[127]
An immunogenic composition according to any of claims 83 to 124 or a vaccine according to claim 125 for use in the treatment or prevention of S. aureus infection in a human subject.
[128]
128. An immunogenic composition for use according to claim 127, wherein the human subject undergoes a surgical procedure, possibly a cardio-thoracic surgery.
[129]
An immunogenic composition for use according to claim 128, wherein the human subject is immunized with a single dose of the immunogenic composition according to any one of claims 83 to 124 or the vaccine of claim 125 at a point from time 5 to 60, 6 to 40, 7 to 30 or 7 to 15 days before undergoing the surgical procedure.
[130]
130. A method of treatment comprising the step of administering to a human subject at risk of developing S. aureus infection an immunologically effective amount of the immunogenic composition of any one of claims 83 to 124 or the vaccine according to claim 125.
[131]
The method of claim 130, wherein the human subject undergoes a surgical procedure, possibly a cardio-thoracic surgery.
[132]
The method of claim 131, wherein the human subject is immunized with a single dose of the immunogenic composition of any one of claims 83 to 124 or the vaccine of claim 125 at a time point of 5 to 60, 6. at 40, 7 to 30 or 7 to 15 days before undergoing the surgical procedure.

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同族专利:

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公开号 | 公开日

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US20160129101A1|2016-05-12|

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HUE051358T2|2021-03-01|

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KR20160018604A|2016-02-17|

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WO2014195280A1|2014-12-11|

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EA201592040A1|2016-06-30|

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AU2014277027A1|2015-12-24|

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MX2015016750A|2016-04-13|

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PL3003363T3|2021-04-06|

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BR112015029931B1|2021-01-12|

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CA2912496A1|2014-12-11|

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CN105517567A|2016-04-20|

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EP3003363A1|2016-04-13|

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EP3003363B1|2020-08-19|

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JP2019123720A|2019-07-25|

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JP2016524619A|2016-08-18|

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US20180104322A1|2018-04-19|

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EA033488B1|2019-10-31|

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SG11201509406QA|2015-12-30|

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GB201310008D0|2013-07-17|

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SI3003363T1|2020-12-31|

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HRP20201683T1|2020-12-25|

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PT3003363T|2020-11-16|

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US20170056490A1|2017-03-02|

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IL242584A|2021-06-30|

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LT3003363T|2020-12-28|

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ES2830785T3|2021-06-04|

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KR102266346B1|2021-06-17|

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DK3003363T3|2020-11-09|

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AU2014277027B2|2017-07-20|

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JP6773830B2|2020-10-21|

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法律状态:
2020-03-27| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190630 |

优先权:

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申请号 | 申请日 | 专利标题

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GB13100086|2013-06-05|

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GBGB1310008.6A|GB201310008D0|2013-06-05|2013-06-05|Immunogenic composition for use in therapy|

---