# Patent

## Title
Process for manufacturing vaccines

## Abstract

The present application discloses a method for making an immunogenic composition comprising an improved way of conducting saccharide-protein conjugation reactions using carbodiimide condensation chemistry. Depending on the nature of the saccharide or protein carrier involved, the quality of the conjugate may be improved by adding one of the reaction components slowly to the reaction mixture. In addition, the conjugate is mixed with a staphylococcal antigen. Immunogenic compositions are further provided comprising the saccharide-protein conjugates made by the methods disclosed.

## Background
This application is filed pursuant to 35 U.S.C. §371 as a United States National Phase Application of International Patent Application Serial No. PCT/EP2008/050011 filed Jan. 2, 2008, which claims priority to Great Britain Application No. 0700136.5 filed in the United Kingdom on Jan. 4, 2007, the content of which is incorporated herein by reference.
The present invention relates to improved methods of making immunogenic compositions by conducting carbodiimide condensation reactions. In particular, it relates to the conjugation of saccharides (particularly staphylococcal saccharides) and proteins using carbodiimide condensation. It also relates to immunogenic compositions that may be made comprising the saccharide-protein conjugates of the invention.
The use of bacterial capsular polysaccharides has been widely used in immunology for many years for the prevention of bacterial disease. A problem with such a use, however, is the T-independent nature of the immune response. These antigens are thus poorly immunogenic in young children. This problem has been overcome through conjugating the polysaccharide antigens to a protein carrier (a source of T-helper epitopes) which may then by used to elicit a T-dependent immune response, even in the first year of life.
Various conjugation techniques are known in the art. Conjugates can be prepared by direct reductive amination methods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508. The conjugation method may alternatively rely on activation of hydroxyl groups of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein. For example, the cyanate ester can be coupled with hexane diamine or adipic acid dihydrazide (ADH or AH) and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the protein carrier. Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094. See also Chu C. et al Infect. Immunity, 1983 245 256.
In general the following types of chemical groups on a protein carrier can be used for coupling/conjugation:
A) Carboxyl (for instance via aspartic acid or glutamic acid) which may be conjugated to natural or derivatised amino groups on saccharide moieties using carbodiimide chemistry;
B) Amino group (for instance via lysine) which may be conjugated to natural or derivatised carboxyl groups on saccharide moieties using carbodiimide chemistry;
C) Sulphydryl (for instance via cysteine);
D) Hydroxyl group (for instance via tyrosine);
E) Imidazolyl group (for instance via histidine);
F) Guanidyl group (for instance via arginine); and
G) Indolyl group (for instance via tryptophan).
On a saccharide, in general the following groups can be used for a coupling: OH, COOH or NH2. Staphylococcal saccharides, for example S. aureus capsular saccharides (such as those form serotypes 5 and/or 8) contain OH and COOH groups. Aldehyde groups can be generated after different treatments known in the art such as: periodate, acid hydrolysis, hydrogen peroxide, etc.
Direct Coupling Approaches:
Saccharide-OH+CNBr or CDAP→cyanate ester+NH2-Prot→conjugate
Saccharide-aldehyde+NH2-Prot→Schiff base+NaCNBH3→conjugate
Saccharide-COOH+NH2-Prot+EDAC→conjugate
Saccharide-NH2+COOH-Prot+EDAC→conjugate
Indirect coupling via spacer (linker) approaches:
Saccharide-OH+CNBr or CDAP→cyanate ester+NH2→NH2→saccharide-NH2+COOH-Prot+EDAC→conjugate
Saccharide-OH+CNBr or CDAP→cyanate ester+NH2-SH→saccharide-SH+SH-Prot (native Protein with an exposed cysteine or obtained after modification of amino groups of the protein by SPDP for instance)→saccharide-S—S-Prot
Saccharide-OH+CNBr or CDAP→cyanate ester+NH2-SH→saccharide-SH+maleimide-Prot (modification of amino groups)→conjugate
Saccharide-COOH+EDAC+NH2-NH2→saccharide-NH2+EDAC+COOH-Prot→conjugate
Saccharide-COOH+EDAC+NH2-SH→saccharide-SH+SH-Prot (native Protein with an exposed cysteine or obtained after modification of amino groups of the protein by SPDP for instance)→saccharide-S—S-Prot
Saccharide-COOH+EDAC+NH2-SH→saccharide-SH+maleimide-Prot (modification of amino groups)→conjugate
Saccharide-Aldehyde+NH2-NH2→saccharide-NH2+EDAC+COOH-Prot→conjugate
As can be observed carbodiimide chemistry (e.g. using EDAC) is very convenient for conjugation reactions as it makes use of groups on the saccharide and/or protein which may be naturally present or easily inserted by derivatisation. It also conveniently links moieties through a peptide bond.
Carbodiimides (RN═C═NR′) are unsaturated compounds with an allene structure (Nakajima and Ikada 1995 Bioconjugate Chem. 6:123-130; Hoare and Koshland 1967 JBC 242:2447-2453). The chemical is relatively unstable at its reaction pH (4.5-6.5), and therefore all components of the saccharide/protein/carbodiimide conjugation reaction tend to be added together in the art.
The present inventors have found that depending on the nature of the saccharide and protein to be conjugated, better characteristics of the final conjugate for vaccine use may be achieved by adding a certain component of the reaction slowly to the mix. In so doing one or more benefits/improvements may be realised such as: saccharide yield in the conjugate, sterile filterability of the conjugate, better control of the conjugation, easier reproducibility, and/or prevention of intra-moiety cross-links.
Accordingly, in one embodiment there is provided a method of making an immunogenic composition comprising a conjugation step of conjugating a saccharide to a protein carrier to make a saccharide-protein conjugate using carbodiimide condensation chemistry, wherein the saccharide comprises (for instance as part of its repeating unit), or has been derivatised to comprise, amino and/or carboxyl groups, and wherein the protein carrier comprises, or has been derivatised to comprise, amino and/or carboxyl groups, comprising the steps of:

    
    
        I)— if the protein carrier comprises both amino and carboxyl groups and the saccharide comprises either amino or carboxyl groups:
        a) mixing the saccharide and aliquot of carbodiimide required to perform the conjugation, and
        b) adding the aliquot of protein carrier required over a period of 35 seconds to 6 hours to form the saccharide-protein conjugate;
        II)— if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises either amino or carboxyl groups:
        a) mixing the protein carrier and aliquot of carbodiimide required to perform the conjugation, and
        b) adding the aliquot of saccharide required over a period of 35 seconds to 6 hours to form the saccharide-protein conjugate; or
        III)— if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises both amino and carboxyl groups:
        a) mixing the protein carrier and saccharide, and
        b) adding the aliquot of carbodiimide required to perform the conjugation over a period of 35 seconds to 6 hours to form the saccharide-protein conjugate;
        and adding a further step to I, II or III of mixing the saccharide-protein conjugate (so formed) with an antigen, for example a staphylococcal antigen.
    
    





DESCRIPTION OF FIGURES
FIG. 1—Polypeptide sequences of preferred proteins. Table 2 provides information on which protein is represented by each SEQ ID.
FIG. 2—Nucleotide sequences encoding preferred proteins. Table 2 provides information on which protein is encoded by each SEQ ID.



DETAILED DESCRIPTION
Any suitable carbodiimide may be used in the conjugation step as long as it is capable of conjugating saccharides and proteins in an aqueous medium. In one embodiment the carbodiimide may be EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) [also known as EDC] or it may be a carbodiimide other than EDAC.
The term “saccharide” throughout this specification may indicate polysaccharide or oligosaccharide and includes both. It may indicate lipopolysaccharide (LPS) or lipooliogosaccharide (LOS). Before use Polysaccharides (such as bacterial polysaccharides) may be isolated from a source strain (e.g. of bacteria) or isolated from the source strain and sized to some degree by known methods (see for example EP497524 and EP497525; Shousun Chen Szu et al.—Carbohydrate Research Vol 152 p7-20 (1986)) for instance by microfluidisation. Polysaccharides can be sized in order to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products. Oligosaccharides have a low number of repeat units (typically 5-30 repeat units) and are typically hydrolysed polysaccharides.
The term “protein carrier” is intended to cover both small peptides and large polypeptides (>10 kDa). Clearly large polypeptides are more likely to contain both reactive amino and carboxyl groups without any modification.
For the purposes of the invention, “native polysaccharide” refers to a saccharide that has not been subjected to a process, the purpose of which is to reduce the size of the saccharide. A polysaccharide can become slightly reduced in size during normal purification procedures. Such a saccharide is still native. Only if the polysaccharide has been subjected to sizing techniques would the polysaccharide not be considered native.
For the purposes of the invention, “sized by a factor up to ×2” means that the saccharide is subject to a process intended to reduce the size of the saccharide but to retain a size more than half the size of the native polysaccharide. ×3, ×4 etc. are to be interpreted in the same way i.e. the saccharide is subject to a process intended to reduce the size of the polysaccharide but to retain a size more than a third, a quarter etc. the size of the native polysaccharide.
By “a further step of mixing the saccharide-protein conjugate with a staphylococcal antigen” it is meant that the saccharide-protein conjugate (which may itself contain a staphylococcal saccharide and/or a staphylococcal protein) is mixed with a further staphylococcal antigen which is not present in the saccharide-protein conjugate.
The 35 second to 6 hour time period in step b) of the method for the addition of the full aliquot of the final component can be 50 seconds to 5 hours, 1 minute to 4 hours, 2 minutes to 3 hours, 3 minutes to 2 hours, 4 to 60 minutes, 5 to 50 minutes, 6 to 40 minutes, 7 to 30 minutes or 8 to 20 minutes. It may be 1 minute to 5 hours, 10 minutes to 4 hours, 20 minutes to 3 hours, 30 minutes to 2 hours, 40 to 90 minutes, or 50 to 70 minutes. This time can be adjusted according to the precise saccharide and protein being conjugated.
In one embodiment the aliquot of the final component of the conjugation step (e.g. of carbodiimide, saccharide or protein) is added to the reaction mixture at a constant rate during the time period (this is conveniently achieved using a pump operating at a constant rate). Alternatively it may be added in stages over the time period. Although this may be done in many ways, in general parts of the aliquot should be added throughout the period. For instance at least one quarter of the aliquot may be added over the first half of the period, and at least one quarter of the aliquot over the second half of the period. The total amount of the aliquot ‘a’ measured, for instance, in mL or mg may be added in 4-100 stages (‘s’) throughout the period. In one embodiment the stages are arranged such that an even amount (a/s) is introduced at all the stages. In one embodiment the stages are evenly spaced throughout the period ‘p’ (in seconds). Thus if one stage takes place at time zero of the period ‘p’, then each subsequent stage could take place at a time which is p/(s−1). The volume of the aliquot of the final component added in step b) may be adjusted in terms of ease of addition of the aliquot to the reaction within the desired time period. The carbodiimide may be added as an aqueous solution (typically buffered at pH 7.5 before being added to the reaction) or as solid powder (EDAC for instance is highly soluble in aqueous media). Of course if the carbodiimide is the last component added to the reaction (situation III step b)), a slow dissolving carbodiimide may be used such that the entire aliquot of powder is added to the reaction all at once but it dissolves at a rate consistent with the desired period over which the aliquot is to be made available to the reaction.
If the protein and/or saccharide has no amino or carboxyl groups (or only has one of these), it may be derivatised to give it one (or to give it the other it does not already have). For instance for a saccharide only comprising reactive hydroxyl groups (e.g. meningococcal serogroup A capsular saccharide), such a group should be used for derivatising on amino or carboxyl groups so that EDAC condensation may be carried out. This may take place within a repeat subunit, or may be a group only present at the end of the saccharide molecule.
It should be noted that where derivatisation takes place, it can be beneficial to only partially derivatise the moiety. For saccharides with repeating subunits, the target epitope may be present in each repeat. Therefore if partial derivatisation takes place (for this it is meant 0.5-20, 1-15, 3-12, or 5-10% of the targeted reactive group is actually derivatised) this can have the benefit of conserving the majority of the epitopes, and preventing too much cross-linking.
If a saccharide or protein already has amino or carboxyl groups only (e.g. Vi saccharide from Salmonella typhi which naturally has carboxyl but not amino groups), derivatisation can take place to give it the other type of group (i.e. amino groups for Vi). It should be noted, however, that as derivatisation can be partial this action can change the preferred reaction of the invention from a type I to a type III. For instance if Vi saccharide is conjugated to a protein carrier comprising both amino and carboxyl groups situation I adds the aliquot of protein slowly in step b). If the Vi saccharide carboxyl group is partially derivatised with amino groups it will have both carboxyl and amino groups, thus situation III adding the aliquot of carbodiimide slowly in step b) becomes most relevant.
Derivatisation may occur through the addition of a hetero- or homo-bifunctional linker. It may take place with similar chemistry as described above for saccharide-protein conjugation step (e.g. CDAP or carbodiimide chemistry). The linker may have between 4 and 20, 4 and 12, or 5 and 10 carbon atoms. It may have two reactive amino groups, two reactive carboxyl groups, or one of each (e.g. hexane diamine, 6-aminocaproic acid, or adipic acid dihydrazide). Typically derivatization takes place through reacting a large excess of the linker with the saccharide and/or protein carrier to be derivatised. This allows derivatization to take place with minimal intra-moiety cross-linking (which otherwise might be possible if for instance a carboxyl group on a saccharide was being derivatised with amino groups using carbodiimide condensation). Excess linker is readily removed using techniques such as diafiltration.
In one embodiment the saccharide comprises a reactive hydroxyl group as part of its repeating unit which is partially derivatised via an amino group on the linker (e.g. with CDAP chemistry). In another embodiment the saccharide comprises a reactive amino group as part of its repeating unit which is partially derivatised via a carboxyl group on the linker (e.g. with carbodiimide chemistry). In a further embodiment the saccharide comprises a reactive carboxyl group as part of its repeating unit which is partially derivatised via an amino group on the linker (e.g. with carbodiimide chemistry).
The aliquot of carbodiimide required to perform the conjugation (whether present in step a) or b) of the reaction of the invention) is 0.01 to 3, 0.05 to 2, or 0.09 to 1 mg carbodiimide/mg saccharide. Although these numbers are calculated in respect of EDAC being the carbodiimide, these numbers may be adjusted if any other carbodiimide is used by multiplying the numbers in the range by: (molecular weight of other carbodiimide)/(molecular weight of EDAC).
In general, the saccharide may be present in the methods of the invention at a final concentration of 0.5-50 mg/ml in step b). This will depend on the size and nature of the saccharide, and the extent of any derivatisation. For instance for oligosaccharides a larger concentration will be required, but for large polysaccharides a much smaller concentration will be more appropriate. If it is towards the high end of partially derivatised with amino or carboxyl groups a smaller concentration may be appropriate to reduce the possibility of any cross-linking. The protein carrier may be present at a final concentration of 1-50 mg/ml in step b).
The initial ratio of protein carrier to saccharide in the methods of the invention can be 5:1 to 1:5, 4:1 to 1:1, or 3:1 to 2:1 (w/w). Again this will depend on the size and nature of the saccharide, and the extent of any derivatisation.
Salt conditions (e.g. NaCl) may also be varied according to the nature of the saccharide/protein. Usually around 0.2M NaCl may be present in step b) of the methods of the invention, but may be 0-2, 0.1-1 or 0.2-0.5 M.
In terms of pH in step b) of the methods of the invention, the reaction pH may be any pH where the carbodiimide is activated—for instance pH 4.5-6.5, 4.7-6.0, or 5-5.5. This pH is typically maintained throughout the reaction by addition of acid/base as required. EDAC is usually stable at pH 7.5, though if the conjugation requires to be done at higher pH compounds which are known to keep the reaction intermediate stable (such as N-hydroxysuccinimide) may also be present in the reaction in step b), in which case the reaction pH in step b) may be maintained at pH 4.5-7.5.
The reaction temperature during step b) of the methods of the invention can be 4-37, 10-32, 17-30, or 22-27° C., and is typically maintained throughout the reaction.
In the methods of the invention, once the entire aliquot has been added in step b) the reaction is typically maintained for a further 10 minutes to 72 hours, 20 minutes to 48 hours, 30 minutes to 24 hours, 40 minutes to 12 hours, 50 minutes to 6 hours, or 1-3 hours. Once the reaction is completed the pH is adjusted to 7.5-9 (towards the higher end of this if N-hydroxysuccinimide is present) to go back to the stable pH range of carbodiimide.
Once conjugated, the saccharide-protein conjugate may be purified from: unreacted components, free saccharide, etc by injecting it on a size exclusion chromatography column (for instance SEPHACRYL® S400HR, Pharmacia). This is typically carried out at 2-8° C. The conjugate may be sterile filtered then stored. Ultimately an effective dose (for instance 1-20, 2-15, or 3-10 μg saccharide/dose) of the saccharide-protein conjugate can be formulated with a pharmaceutically acceptable excipient (for instance a salt or adjuvant) to manufacture an immunogenic composition or vaccine.
In terms of the saccharides of the invention, any saccharide of viral, fungal, bacterial or eukaryotic source may be conjugated using the conjugation step of the methods of the invention. It may be the Vi saccharide from Salmonella typhi, or a saccharide other than Vi. It may be the capsular saccharide Hib from H. influenzae type b, or may be a saccharide other than Hib. In one embodiment the saccharide is a bacterial capsular saccharide, for instance derived from a bacterium selected from a list consisting of: N. meningitidis serogroup A (MenA), B (MenB), C (MenC), W135 (MenW) or Y (MenY), Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F, Group B Streptococcus group Ia, Ib, II, III, IV, V, VI, or VII, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Salmonella typhi (Vi saccharide), Vibrio cholerae, or H. influenzae type b.
The weight-average molecular weight of the saccharide may be 1000-2000000, 5000-1000000, 10000-500000, 50000-400000, 75000-300000, or 100000-200000. The molecular weight or average molecular weight of a saccharide herein refers to the weight-average molecular weight (Mw) of the saccharide measured prior to conjugation and is measured by MALLS. The MALLS technique is well known in the art and is typically carried out as described in example 2. For MALLS analysis of saccharides, two columns (TSKG6000 and 5000PWxl) may be used in combination and the saccharides are eluted in water. Saccharides are detected using a light scattering detector (for instance Wyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498 nm). In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or 1-1.05 and after conjugation to a carrier protein, the polydispersity of the conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are by MALLS.
The saccharide may be either a native polysaccharide or may have been sized by a factor of no more than 2, 4, 6, 8, 10 or 20 fold (for instance by microfluidization [e.g. by EMULSIFLEX® C-50 apparatus] or other known technique [for instance heat, chemical, oxidation, sonication methods]). Oligosaccharides may have been sized substantially further [for instance by known heat, chemical, or oxidation methods].
The structures of most of these saccharides are known (and therefore whether they naturally have any amino or carboxyl groups for carbodiimide chemistry, or any other reactive group which may be derivatised with amino or carboxyl groups (see table 1 below).











TABLE 1







Natural NH2
Natural COOH
Other reactive



group
group
group



















S. aureus





PS5
No
Yes
OH


PS8
No
Yes
OH


N. meningitidis


MenA
No
No
OH


MenC
No
Yes
OH


MenW135
No
Yes
OH


MenY
No
Yes
OH


MenB
No (can be
Yes
OH/N-propyl



generated if



de-N-acetylated)


Gp. B Streptococcus


Ia, Ib
No
Yes
OH


II
No
Yes
OH


III
No
Yes
OH


IV
No
Yes
OH


V
No
Yes
OH


VI
No
Yes
OH


VII
No
Yes
OH


S. typhi


Vi
No
Yes
No


S. pneumoniae


PS1
Yes
Yes
OH


PS3, 4, 5, 8, 9, 12F
No
Yes
OH


Vibrio cholorea


Capsular saccharide
yes
No
OH


H. influenzae B Hib
No
No
OH


LOS


Nmen/Mcat/Hi
Yes on PEA
Yes on KDO
OH









The protein carrier may be any peptide or protein. In an embodiment it is a staphylococcal protein, optionally selected from the staphylococcal proteins listed below. It may comprise one or more T-helper epitopes. In one embodiment of the invention the protein carrier is selected from the group consisting of: TT, DT, CRM197, fragment C of TT, protein D of H. influenzae, pneumococcal PhtD, and pneumococcal Pneumolysin. The carrier protein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin [note all such variants of TT are considered to be the same type of carrier protein for the purposes of this invention], diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711] (note all such variants of DT are considered to be the same type of carrier protein for the purposes of this invention), pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), OMPC (meningococcal outer membrane protein—usually extracted from N. meningitidis serogroup B—EP0372501), synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761), H. influenzae Protein D (EP594610 and WO 00/56360), pneumococcal PhtA (WO 98/18930, also referred to Sp36), pneumococcal PhtD (disclosed in WO 00/37105, and is also referred to Sp036D), pneumococcal PhtB (disclosed in WO 00/37105, and is also referred to Sp036B), or PhtE (disclosed in WO00/30299 and is referred to as BVH-3).
The method of the invention includes a further step of mixing the saccharide-protein conjugate with an antigen, for example a staphylococcal antigen. A staphylococcal antigen may be chosen from the antigens described below, although this list is not exclusive of other antigens derived from staphylococci. In an embodiment, the staphylococcal antigen is a saccharide, teichoic acid or lipoteichoic acid (LTA), any of which is/are optionally conjugated, optionally using a conjugation method described herein. In an embodiment, the staphylococcal antigen is a protein, optionally as described herein.
Capsular Saccharides from S. aureus 
In an embodiment, the method of the invention comprises the step of mixing the saccharide-protein conjugate of the invention with S. aureus capsular saccharides (for example capsular saccharides from S. aureus type 5 and/or type 8). In a further embodiment, the method of the invention conjugates a S. aureus capsular saccharide (for example capsular saccharides from S. aureus type 5 and/or type 8) to a protein a make a saccharide-protein conjugate according to the invention.
Most strains of S. aureus that cause infection in man contain either Type 5 or Type 8 polysaccharides. Approximately 60% of human strains are Type 8 and approximately 30% are Type 5. The structures of Type 5 and Type 8 capsular polysaccharide antigens are described in Moreau et al Carbohydrate Res. 201; 285 (1990) and Fournier et al Infect. Immun. 45; 87 (1984). Both have FucNAcp in their repeat unit as well as ManNAcA which can be used to introduce a sulfhydryl group.
Recently (Jones Carbohydrate Research 340, 1097-1106 (2005)) NMR spectroscopy revised the structures of the capsular polysaccharides to:
Type 5
→4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→
Type 8
→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→
Saccharides may be extracted from the appropriate strain of S. aureus using method well known to the skilled man, for instance as described in U.S. Pat. No. 6,294,177. For example, ATCC 12902 is a Type 5 S. aureus strain and ATCC 12605 is a Type 8 S. aureus strain.
Saccharides are of native size or alternatively may be sized, for instance by microfluidisation, ultrasonic irradiation or by chemical treatment. The invention also covers oligosaccharides derived from the type 5 and 8 polysaccharides from S. aureus. 
The weight-average molecular weight of the saccharide may be 1000-2000000, 5000-1000000, 10000-500000, 50000-400000, 75000-300000, or 100000-200000. The molecular weight or average molecular weight of a saccharide herein refers to the weight-average molecular weight (Mw) of the saccharide measured prior to conjugation and is measured by MALLS. The MALLS technique is well known in the art and is typically carried out as described in example 2. For MALLS analysis of saccharides, two columns (TSKG6000 and 5000PWxl) may be used in combination and the saccharides are eluted in water. Saccharides are detected using a light scattering detector (for instance Wyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498 nm). In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or 1-1.05 and after conjugation to a carrier protein, the polydispersity of the conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are by MALLS.
The type 5 and/or 8 capsular polysaccharide or oligosaccharides included in the process or immunogenic composition of the invention are optionally O-acetylated. In an embodiment, the degree of O-acetylation of type 5 capsular polysaccharide or oligosaccharide is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%. 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In an embodiment, the degree of O-acetylation of type 8 capsular polysaccharide or oligosaccharide is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%. 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In an embodiment, the degree of O-acetylation of type 5 and type 8 capsular polysaccharides or oligosaccharides is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%. 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In an embodiment, the type 5 and/or 8 capsular saccharides are de-O-acetylated.
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 (Lernercinier and Jones 1996, Carbohydrate Resarch 296; 83-96, WO 05/033148 or WO 00/56357).
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 1-8 hours. In order to maintain high levels of O-acetylation on type 5 and/or 8 polysaccharide or oligosaccharide, treatments which would lead to hydrolysis of the O-acetyl groups are minimised. For example treatment at extremes of pH are minimised.
In an embodiment, the O-acetylated staphylococcal saccharides as described above are conjugated using the conjugation step of the method of the invention and is mixed with a further staphylococcal antigen in the method of the invention.
Poly N-Acetylated Glucosamine (PNAG)
In an embodiment, the method of the invention comprises the step of mixing the saccharide-protein conjugate of the invention with poly N-acetylated glucosamine (PNAG) antigen.
PNAG is a polysaccharide intercellular adhesin and is composed of a polymer of β-(1→6)-linked glucosamine, optionally substituted with N-acetyl and O-succinyl constituents. This polysaccharide is present in both S. aureus and S. epidermidis and can be isolated from either source (Joyce et al 2003, Carbohydrate Research 338; 903; Maira-Litran et al 2002, Infect. Imun. 70; 4433). For example, PNAG may be isolated from S. aureus strain MN8m (WO 04/43407).
The polysaccharide previously known as poly-N-succinyl-β-(1→6)-glucosamine (PNSG) was recently shown not to have the expected structure since the identification of N-succinylation was incorrect (Maira-Litran et al 2002, Infect. Imun. 70; 4433). Therefore the polysaccharide formally known as PNSG and now found to be PNAG is also encompassed by the term PNAG.
PNAG may be of different sizes varying from over 400 kDa to between 75 and 400 kDa to between 10 and 75 kDa to oligosaccharides composed of up to 30 repeat units (of β-(1→6)-linked glucosamine, optionally substituted with N-acetyl and O-succinyl constituents). Any size of PNAG polysaccharide or oligosaccharide may be use in an immunogenic composition of the invention, for example a size of over 40 kDa can be used. Sizing may be achieved by any method known in the art, for instance by microfluidisation, ultrasonic irradiation or by chemical cleavage (WO 03/53462, EP497524, EP497525).
Size ranges of PNAG are for example 40-400 kDa, 50-350 kDa, 40-300 kDa, 60-300 kDa, 50-250 kDa and 60-200 kDa.
PNAG can have different degree of acetylation due to substitution on the amino groups by acetate. PNAG produced in vitro is almost fully substituted on amino groups (95-100%). Alternatively, a deacetylated PNAG can be used having less than 50%, 40%, 30%, 20%, 10% or 5% N-acetylation. Use of a deacetylated PNAG allows opsonic killing of Gram positive bacteria, preferably S. aureus and/or S. epidermidis (WO 04/43405). In an embodiment, the PNAG has a size between 40 kDa and 300 kDa and is deacetylated so that less than 50%, 40%, 30%, 20%, 10% or 5% of amino groups are N acetylated.
In an embodiment, the PNAG is not O-succinylated or is O-succinylated on less than 25, 20, 15, 10, 5, 2, 1 or 0.1% of residues.
The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide or oligosaccharide in which less than 50%, 40%, 30%, 20%, 10% or 5% of the amino groups are acetylated.
As used herein, the term PNAG encompasses both acetylated and deacetylated forms of the saccharide.
In an embodiment, PNAG is deacetylated to form dPNAG by chemically treating the native polysaccharide. For example, the native PNAG is treated with a basic solution such that the pH rises to above 10. For instance the PNAG is treated with 0.1-5M, 0.2-4M, 0.3-3M, 0.5-2M, 0.75-1.5M or 1M NaOH, KOH or NH4OH. Treatment is for at least 10 or 30 minutes, or 1, 2, 3, 4, 5, 10, 15 or 20 hours at a temperature of 20-100, 25-80, 30-60 or 30-50 or 35-45° C. dPNAG may be prepared as described in WO 04/43405.
In an embodiment, the polysaccharide(s) included in the process of the invention are conjugated to a carrier protein as described below or alternatively unconjugated.
S. aureus 336 Antigen
In an embodiment, the method of the invention comprises the step of mixing the saccharide-protein conjugate of the invention with S. aureus 336 antigen (as described in U.S. Pat. No. 6,294,177).
The 336 antigen comprises β-linked hexosamine, contains no O-acetyl groups and specifically binds to antibodies to S. aureus Type 336 deposited under ATCC 55804.
In an embodiment, the 336 antigen is a polysaccharide which is of native size or alternatively may be sized, for instance by microfluidisation, ultrasonic irradiation or by chemical treatment. The invention also covers oligosaccharides derived from the 336 antigen.
The 336 antigen, where included in the process of the invention is preferably conjugated to a carrier protein as described below or are alternatively unconjugated.
Strains ATCC-31432, SE-360 and SE-10 of S. epidermidis are characteristic of three different capsular types, I, II and III respectively (Ichiman and Yoshida 1981, J. Appl. Bacteriol. 51; 229). Capsular polysaccharides extracted from each serotype of S. epidermidis constitute Type I, II and III polysaccharides. Polysaccharides may be extracted by serval methods including the method described in U.S. Pat. No. 4,197,290 or as described in Ichiman et al 1991, J. Appl. Bacteriol. 71; 176.
In one embodiment of the invention, the process comprises mixing the saccharide-protein conjugate with type I and/or II and/or III polysaccharides or oligosaccharides from S. epidermidis. 
Polysaccharides are of native size or alternatively may be sized, for instance by microfluidisation, ultrasonic irradiation or chemical cleavage. The invention also covers oligosaccharides extracted from S. epidermidis strains.
These polysaccharides are unconjugated or are preferably conjugated as described herein.
Conjugation of Polysaccharides
Amongst the problems associated with the use of polysaccharides in vaccination, is the fact that polysaccharides per se are poor immunogens. Strategies, which have been designed to overcome this lack of immunogenicity, include the linking of the polysaccharide to large protein carriers, which provide bystander T-cell help. It is preferred that the polysaccharides utilised in the invention are linked to a protein carrier which provide bystander T-cell help. Examples of these carriers which may be used for coupling to polysaccharide or oligosaccharide immunogens include the Diphtheria and Tetanus toxoids (DT, DT Crm197 and TT), Keyhole Limpet Haemocyanin (KLH), Pseudomonas aeruginosa exoprotein A (rEPA) and the purified protein derivative of Tuberculin (PPD), protein D from Haemophilus influenzae, pneumolysin or fragments of any of the above. Fragments suitable for use include fragments encompassing T-helper epitopes. In particular protein D fragment will preferably contain the N-terminal 1⁄3 of the protein. Protein D is an IgD-binding protein from Haemophilus influenzae (EP 0 594 610 B1).
An alternative carrier protein to use in the processes of the invention is a single staphylococcal protein or fragment thereof or a fusion protein comprising at least or exactly 1, 2, 3 or 4 or more of the staphylococcal proteins or fragments thereof listed in the section below.
A new carrier protein that would be particularly advantageous to use in the context of a staphylococcal vaccine is staphylococcal alpha toxoid. The native form may be conjugated to a polysaccharide since the process of conjugation reduces toxicity. Preferably a genetically detoxified alpha toxin such as the His35Leu or His 35 Arg variants are used as carriers since residual toxicity is lower. Alternatively the alpha toxin is chemically detoxified by treatment with a cross-linking reagent, formaldehyde or glutaraldehyde. A genetically detoxified alpha toxin is optionally chemically detoxified, preferably by treatment with a cross-linking reagent, formaldehyde or glutaraldehyde to further reduce toxicity.
Proteins
The method of the invention optionally comprises a step of mixing the saccharide-protein conjugate of the invention with a staphylococcal protein, for example a protein from S. aureus or S. epidermidis. Some embodiments of the invention contain proteins from both S. aureus and S. epidermidis. 
In an independent embodiment of the method of the invention, a staphylococcal protein is used as the protein carrier to which the saccharide is conjugated in the method of the invention.

    
    
        Accordingly the invention provides a method of conjugating a saccharide to a staphylococcal protein carrier using carbodiimide condensation chemistry, wherein the saccharide comprises (for instance as part of its repeating unit), or has been derivatised to comprise, amino and/or carboxyl groups, and wherein the protein carrier comprises, or has been derivatised to comprise, amino and/or carboxyl groups, comprising the steps of:
        
            I)— if the staphylococcal protein carrier comprises both amino and carboxyl groups and the saccharide comprises either amino or carboxyl groups:
            a) mixing the saccharide and aliquot of carbodiimide required to perform the conjugation, and
            b) adding the aliquot of staphylococcal protein carrier required over a period of 35 seconds to 6 hours;
            II)— if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises either amino or carboxyl groups:
            a) mixing the staphylococcal protein carrier and aliquot of carbodiimide required to perform the conjugation, and
            b) adding the aliquot of saccharide required over a period of 35 seconds to 6 hours;
            III)— if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises both amino and carboxyl groups:
            a) mixing the staphylococcal protein carrier and saccharide, and
            b) adding the aliquot of carbodiimide required to perform the conjugation over a period of 35 seconds to 6 hours.
        
        
    
    


In an embodiment, the methods of the invention use an isolated protein which comprises an amino acid sequence which has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of any sequence of FIG. 1.
Where a protein is specifically mentioned herein, it is optionally a reference to a native or recombinant, full-length protein or optionally a mature protein in which any signal sequence has been removed. The protein may 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, preferably 20 amino acids, more preferably 30 amino acids, more preferably 40 amino acids or 50 amino acids, most preferably 100 amino acids, taken contiguously from the amino acid sequence of the protein. In addition, such immunogenic fragments are typically immunologically reactive with antibodies generated against the Staphylococcal proteins or with antibodies generated by infection of a mammalian host with Staphylococci or contain T cell epitopes. Immunogenic fragments also includes fragments that when administered at an effective dose, (either alone or as a hapten bound to a carrier), elicit a protective immune response against Staphylococcal infection, optionally it is protective against S. aureus and/or S. epidermidis infection. Such an immunogenic fragment may include, for example, the protein lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a C-terminal anchor domain. In an embodiment, the immunogenic fragment according to the invention comprises substantially all of the extracellular domain of a protein which has at least 85%, 90%, 95%, 97% or 99% identity, to that a sequence selected from FIG. 1 over the entire length of the fragment sequence.
In an embodiment, the methods of the invention may use fusion proteins of Staphylococcal proteins, or fragments of staphylococcal proteins. Such fusion proteins may be made recombinantly and may comprise one portion of at least 2, 3, 4, 5 or 6 staphylococcal proteins. Alternatively, a fusion protein may comprise multiple portions of at least 2, 3, 4 or 5 staphylococcal proteins. These may combine different Staphylococcal proteins or fragments thereof in the same protein. Alternatively, the invention also includes individual fusion proteins of Staphylococcal proteins or fragments thereof, as a fusion protein with heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: β-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), epitope tags such as FLAG, myc tag, poly histidine, or viral surface proteins such as influenza virus haemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM197.
Proteins
In an embodiment, the methods of the invention use one or more of the proteins mentioned below. Many of the proteins fall into the categories of extracellular component binding proteins, transporter proteins or toxins and regulators of virulence. The methods of the invention optionally use a staphylococcal extracellular component binding protein or a staphylococcal transporter protein or a staphylococcal toxin or regulator of virulence. The methods of the invention optionally use at least or exactly 1, 2, 3, 4, 5 or 6 staphylococcal proteins.
The following table (table 2) sets out the SEQ ID numbers of protein sequences and DNA sequences that are found in FIG. 1 and FIG. 2 respectively. SA indicates a sequence from S. aureus and SE indicates a sequence from S. epidermidis.









TABLE 2





Name
Protein sequence
DNA sequence







Immunodominant ABC transporter




SA
SEQ ID 1
SEQ ID 34


SE
SEQ ID 2
SEQ ID 35


Laminin receptor


SA
SEQ ID 3
SEQ ID 36


SE
SEQ ID 4
SEQ ID 37


Secretory Antigen A SsaA


SA 1
SEQ ID 5
SEQ ID 38


SA 2
SEQ ID 6
SEQ ID 39


SE
SEQ ID 7
SEQ ID 40


SitC


SA
SEQ ID 8
SEQ ID 41


SE
SEQ ID 9
SEQ ID 42


IsaA/PisA (IssA)


SA
SEQ ID 10
SEQ ID 43


SE
SEQ ID 11
SEQ ID 44


EbhA/B


SA EbhA
SEQ ID 12
SEQ ID 45


SA EbhB
SEQ ID 13
SEQ ID 46


SE EbhA
SEQ ID 14
SEQ ID 47


SE EbhB
SEQ ID 15
SEQ ID 48


Accumulation-assoc pro Aap


SA
SEQ ID 16
SEQ ID 49


SE
SEQ ID 17
SEQ ID 50


RNA III activating protein RAP


SA
SEQ ID 18
SEQ ID 51


SE
SEQ ID 19
SEQ ID 52


FIG/SdrG


SA
SEQ ID 20
SEQ ID 53


SE
SEQ ID 21
SEQ ID 54


Elastin binding protein EbpS


SA
SEQ ID 22
SEQ ID 55


SE
SEQ ID 23
SEQ ID 56


Extracellular protein EFB SA
SEQ ID 24
SEQ ID 57


alpha toxin SA
SEQ ID 25
SEQ ID 58


SBI SA
SEQ ID 26
SEQ ID 59


IsdA SA
SEQ ID 27
SEQ ID 60


IsdB SA
SEQ ID 28
SEQ ID 61


SdrC SA
SEQ ID 29
SEQ ID 62


ClfA SA
SEQ ID 30
SEQ ID 63


FnbA SA
SEQ ID 31
SEQ ID 64


ClfB SA
SEQ ID 32
SEQ ID 65


Coagulase SA
SEQ ID 33
SEQ ID 66


FnbB SA
SEQ ID 67
SEQ ID 77


MAP SA
SEQ ID 68
SEQ ID 78


HarA SA
SEQ ID 69
SEQ ID 79


Autolysin glucosaminidase SA
SEQ ID 70
SEQ ID 80


Autolysin amidase SA
SEQ ID 71
SEQ ID 81


Ebh fragment SA
SEQ ID 72
SEQ ID 82


Autolysin Ant SA
SEQ ID 73
SEQ ID 83


SdrC SA
SEQ ID 74
SEQ ID 84


MRPII SA
SEQ ID 75
SEQ ID 85


SdrG SA
SEQ ID 76
SEQ ID 86









Extracellular Component Binding Proteins
Extracellular component binding proteins are proteins that bind to host extracellular components. The term includes, but is not limited to adhesins.
Examples of extracellular component binding proteins include laminin receptor (Naidu et al J. Med. Microbiol. 1992, 36; 177), Protein A, SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234, Wiltshire and Foster Infec. Immun. 2001, 69; 5198), EbhA (Williams et al Infect. Immun. 2002, 70; 6805), EbhB, Elastin binding protein (EbpS) (Park et al 1999, J. Biol. Chem. 274; 2845), EFB (FIB) (Wastfelt and Flock 1995, J. Clin. Microbiol. 33; 2347), SBI (Zhang et al FEMS Immun. Med. Microbiol. 2000, 28; 211), protein A (PCT/EP2006/069944), autolysin (Rupp et al 2001, J. Infect. Dis. 183; 1038), ClfA (U.S. Pat. No. 6,008,341, McDevitt et al Mol. Microbiol. 1994, 11; 237), SdrC (WO 99/27109), SdrD (WO 99/27109), SdrE (WO 99/27109), SdrG (McCrea et al Microbiology 2000, 146; 1535), SdrH (McCrea et al Microbiology 2000, 146; 1535), Lipase GehD (US2002/0169288), SasA (WO 06/121664, Mazmanian et al Molecular Microbiology 40; 1049, 2001 and WO 06/121664), FnbA (Flock et al Mol Microbiol. 1994, 12; 599, U.S. Pat. No. 6,054,572), FnbB (WO 97/14799, Booth et al 2001 Infec. Immun. 69; 345), collagen binding protein Cna (Visci et al 2000, J. Biol. Chem. 275; 39837), ClfB (WO 99/27109), FbpA (Phonimdaeng et al 1988 J. Gen Microbiol. 134; 75), Npase (Flock 2001 J. Bacteriol. 183; 3999), IsaA/PisA (Lonenz et al FEMS Immuno. Med. Microbiol. 2000, 29; 145), SsaA (Lang et al FEMS Immunol. Med. Microbiol. 2000, 29; 213), EPB (Hussain and Hermann symposium on Staph Denmark 14-17th 2000), SasH (Robertson et al Antimicrobial agents and chemotherapy 47; 3926, 2003) SSP-1 (Veenstra et al 1996, J. Bacteriol. 178; 537), SSP-2 (Veenstra et al 1996, J. Bacteriol. 178; 537), 17 kDa heparin binding protein HBP (Fallgren et al 2001, J. Med. Microbiol. 50; 547), Vitronectin binding protein (Li et al 2001, Curr. Microbiol. 42; 361), fibrinogen binding protein, coagulase, Fig (WO 97/48727) and MAP (U.S. Pat. No. 5,648,240)
SitC/MntC/Saliva Binding Protein
This is an ABC transporter protein which is a homologue of adhesin PsaA in S. pneumoniae. It is a highly immunogenic 32 kDa lipoprotein which is distributed through the bacterial cell wall (Cockayne et al Infect. Immun. 1998 66; 3767). It is expressed in S. aureus and S. epidermidis as a 32 kDa lipoprotein and a 40 kDa homologue is present in S. hominis. In S. epidermidis, it is a component of an iron-regulated operon. It shows considerable homology to both adhesins including FimA of Streptococcus parasanguis, and with lipoproteins of a family of ABC transporters with proven or putative metal iron transport functions. Therefore SitC is included as an extracellular biding protein and as a metal ion transporter.
The saliva binding protein disclosed in U.S. Pat. No. 5,801,234 is also a form of SitC and can be included in an immunogenic composition of the invention.
ClfA and ClfB
Both these proteins have fibrinogen binding activity and trigger S. aureus to form clumps in the presence of plasma. They contain a LPXTG (SEQ ID NO: 102) motif common to wall associated proteins.
ClfA is described in U.S. Pat. No. 6,008,341 and ClfB is described in WO 99/27109.
Coagulase (FbpA)
This is a fibrinogen binding protein which triggers S. aureus to form clumps in the presence of plasma. It is described in references related to Coagulase: Phonimdaeng et al (J. Gen. Microbio. 1988, 134:75-83), Phonimdaeng et al. (Mol Microbiol 1990; 4:393-404), Cheung et al. (Infect Immun 1995; 63:1914-1920) and Shopsin et al. (J. CLin. Microbiol. 2000; 38:3453-3456).
Preferred fragments for inclusion in the immunogenic composition of the invention include the mature protein in which the signal peptide has been removed (amino acids 27 to the C-terminus).
Coagulase has three distinct domains. Amino acids 59-297 which is a coiled coil region, amino acids 326-505 which is a proline and glycine rich region and the C-terminal domain from amino acid 506 to 645 which has a beta sheet conformation. Each of these domains is a fragment which may be incorporated into the immunogenic composition of the invention.
SdrG
This protein is described in WO 00/12689. SdrG is found in coagulase negative staphylococci and is a cell wall associated protein containing a LPXTG (SEQ ID NO: 102) sequence.
SdrG contains a signal peptide (amino acids 1-51), a region containing fibrinogen binding sites and collagen binding sites (amino acids 51-825), two CnaB domains (amino acids 627-698 and 738-809), a SD repeat region (amino acids 825-1000) and an anchor domain (amino acids 1009-1056).
Preferred fragments of SdrG include polypeptides in which the signal peptide and/or the SD repeats and the anchor domain have been removed. These include polypeptides comprising or consisting of amino acids 50-825, amino acids 50-633, amino acids 50-597 (SEQ ID NO 2 of WO 03/76470), amino acids 273-597 (SEQ ID NO 4 of WO 03/76470), amino acids 273-577 (SEQ ID NO 6 of WO 03/76470) amino acids 1-549, amino acids 219-549, amino acids 225-549, amino acids 219-528, amino acids 225-528 of SEQ ID NO: 70 or 20 or 21.
Preferably, an SdrG polypeptide having a sequence at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% homologous to the sequence of SEQ ID NO: 70, 20 or 21 is incorporated into the immunogenic composition of the invention.
The compositions of the invention optionally comprise a fragment of the SdrG polypeptides described above.
In an embodiment fragments have the signal peptide and/or the SD repeat domain and/or the anchoring domain deleted. For example sequences corresponding to amino acids 1-713, 1-549, 225-549, 225-529, 24-717, 1-707, 1-690, 1-680, 1-670, 1-660, 1-650, 1-640, 1-630, 1-620, 1-610, 1-600, 34-707, 44-697, 36-689 of SEQ ID 70 or sequences having 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID 70 or 20 or 21.
In an embodiment, fragments with the signal peptide deleted have a methionine residue at the N-terminus of the fragment to ensure correct translation.
In an embodiment, the fragment has the following sequence (SEQ ID NO:87):—







MEENSVQDVKDSNTDDELSDSNDQSSDEEKNDVINNNQSINTDDNNQIIK


 


KEETNNYDGIEKRSEDRTESTTNVDENEATFLQKTPQDNTHLTEEEVKES


 


SSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESN


 


TESGKEENTIEQPNKVKEDSTTSQPSGYTNIDEKISNQDE


 


LLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQ


 


SITEGYDDSEGVIKAHDAENLIYDVTFEVDDKVKSGDTMTVDIDKNTVPS


 


DLTDSFTIPKIKDNSGEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLK


 


LTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSM


 


FTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGST


 


IIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNI


 


NFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYD


 


NTIAFSTSSGQGQGDLPPEKTYKIGDYVWEDVDKDGIQNTNDNEKPLSNV


 


LVTLTYPDGTSKSVRTDEDGKYQFDGLKNGLTYKITFETPEGYTPTLKHS


 


GTNPALDSEGNSVWVTINGQDDMTIDSGFYQTPKYSLGNY


 


VWYDTNKDGIQGDDEKGISGVKVTLKDENGNIISTTTTDENGKYQFDNLN


 


SGNYIVHFDKPSGMTQTTTDSGDDDEQDADGEEVHVTITDHDDFSIDNGY


 


YDDE






EbhA and EbhB
EbhA and EbhB are proteins that are expressed in both S. aureus and S. epidermidis (Clarke and Foster Infect. Immun. 2002, 70; 6680, Williams et al Infect. Immun. 2002, 20; 6805) and which bind to fibronectin. Since fibronectin is an important component of extracellular matrix, EbhA and EbhB have an important function in adhering staphylococci to host extracellular matrix.
The Ebh proteins are large, having a molecular weight of 1.1 megadaltons. It is advantageous to use a fragment of the Ebh protein rather than the complete sequence due to ease of production and formulation. The central region of the protein contains imperfect repeats which contain fibronectin binding sites. Fragments containing one or more of the repeat domains described below are preferred fragments for incorporation into the immunogenic composition of the invention.
Ebh proteins contain imperfect repeats units of 127 amino acids in length which are characterised by containing the consensus sequence:—







L.G.{10}A.{13}Q.{26}L...M..L.{33}A


or


 


.{19}L.G.{10}A.{13}Q.{26}L...M..L.{33}A.{12}


or


 


.....I/V..A...I/V..AK.ALN/DG..NL..AK..A.{6}L..LN.A


QK..L..QI/V..A..V..V.{6}A..LN/D.AM..L...I/V.D/E...


TK.S.NY/F.N/DAD..K..AY/F..AV..A..I/V.N/D.......


 


Where ‘.’ means any amino acid and ‘.{10}’ means


any 10 amino acids and I/V indicates alternative


choices of amino acid.






By reference to the sequence disclosed in Kuroda et al (2001) Lancet 357; 1225-1240, and Table 3, the repeat sequences within Ebh proteins are readily deduced.
In an embodiment, fragments to be included in the immunogenic composition of the invention include proteins containing of one, two, three, four, five, six, seven, eight, nine, ten or more than 10 of the 127 amino acid repeat units. Such fragments may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the 127 amino acid repeat region or may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats with additional amino acid residues present at either or both ends of the fragment. Optionally the fragment is the H2 polypeptide of about 44 kDa spaning three repeats (amino acids 3202-3595) as described in Clarke et al Infection and Immunity 70, 6680-6687, 2002. Such fragments will preferably be able to bind fibronectin and/or to elicit antibodies that are reactive against the whole Ebh protein.
The Ebh proteins are capable of binding to fibronectin. Preferred fragments of these polypeptides sequences retain the ability to bind to fibronectin. Binding to fibronectin can be assessed by ELISA as described by Clarke et al (Infection and Immunity 70; 6680-6687 2002).
In an embodiment, the fragment is one which comprises a B-cell or T-helper epitope, for example those fragments/peptides described in Tables 4 and 5.







TABLE 3







Repeat sequences in the full-length sequence of Ebh.


The full-length sequence of Ebh is disclosed in Kuroda et al (2001)


Lancet 357; 1225-1240. The following table shows the amino acid


residues at which the 127 amino acid repeats begin and end


within the full length sequence.










Begin
End













1
3204
3330


2
3331
3457


3
3457
3583


4
3583
3709


5
3709
3835


6
3835
3961


7
3961
4087


8
4200
4326


9
4326
4452


10
4452
4578


11
4578
4704


12
4704
4830


13
4830
4956


14
4956
5082


15
5082
5208


16
5208
5334


17
5334
5460


18
5460
5586


19
5585
5711


20
5711
5837


21
5837
5963


22
5963
6089


23
6089
6215


24
6215
6341


25
6341
6467


26
6467
6593


27
6593
6719


28
6719
6845


29
6845
6971


30
6971
7097


31
7097
7223


32
7223
7349


33
7349
7475


34
7475
7601


35
7601
7727


36
7727
7853


37
7852
7978


38
7978
8104


39
8104
8230


40
8230
8356


41
8356
8482


42
8482
8608


43
8604
8730


44
8858
8984
















TABLE 4





B-cell epitope prediction for a 127 amino acid


repeat:















The full-length sequence is disclosed in Kuroda


et al (2001) Lancet 357; 1225-1240. One of


these repeats, encoded by amino acids 3204-3331


of the full-length sequence was chosen to carry


out an epitope prediction:-


 


MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNA


LTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNA


DTNKKNDYNNAYNHANDIINGNAQHPVI
















Begin
End
Epitope sequence
Start
Stop

















5
10
TVNQKA
3208
3213



 



14
19
KSTKDA
3217
3222



 



21
33
DGQQNLQRAKTEA
3224
3236



 



42
51
DLNQAQKNAL
3245
3254



 



66
74
DIKQTTQSL
3269
3277



 



100
112
ADTNKKNDYNNAY
3303
3315



 



117
123
DIINGNA
3320
3326







The “Begin” and “End” columns present the position of the predicted B-cell epitopes in the 127 amino acid repeat



The “Start” and “Stop” columns present the position of the predicted B-cell epitopes in the Ebh full length sequence













TABLE 5





T-helper cell epitope prediction in Ebh:















The full-length sequence is disclosed in TrEMBL


database, sequence reference Q8NWQ6. One of these


repeats, encoded by amino acids 3204-3331 of the


full-length sequence was chosen to carry out an


epitope prediction:-


 


MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNA


LTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNA


DTNKKNDYNNAYNHANDIINGNAQHPVI












Position

Position


repeat
Epitope sequence
sequence












1
MDVNTVNQK
3204


 


3
VNTVNQKAA
3206


 


6
VNQKAASVK
3209


 


26
LQRAKTEAT
3229


 


37
ITHASDLNQ
3240


 


43
LNQAQKNAL
3246


 


51
LTQQVNSAQ
3254


 


55
VNSAQNVHA
3258


 


61
VHAVNDIKQ
3264


 


64
VNDIKQTTQ
3267


 


67
IKQTTQSLN
3270


 


74
LNTAMTGLK
3277


 


78
MTGLKRGVA
3281


 


81
LKRGVANHN
3284


 


85
VANHNQVVQ
3288


 


91
VVQSDNYVN
3294


 


92
VQSDNYVNA
3295


 


97
YVNADTNKK
3301


 


98
VNADTNKKN
3302


 


108
YNNAYNHAN
3311


 


112
YNHANDIIN
3315


 


118
IINGNAQHP
3321


 


119
INGNAQHPV
3322





The “Position repeat” column presents the position of the predicted T-cell epitopes in the repeat


The “Position sequence” column presents the position of the predicted T-cell epitopes in the Ebh full length sequence






Fragments of the proteins of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these fragments may be employed as intermediates for producing the full-length proteins of the invention.
In an embodiment, variants are used in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
Elastin Binding Protein (EbpS)
EbpS is a protein containing 486 amino acids with a molecular weight of 83 kDa. It is associated with the cytoplasmic membrane of S. aureus and has three hydrophobic regions which hold the protein in the membrane (Downer et al 2002, J. Biol. Chem. 277; 243; Park et al 1996, J. Biol. Chem. 271; 15803).
Two regions between amino acids 1-205 and 343-486 are surface exposed on the outer face of the cytoplasmic membrane. The ligand binding domain of EbpS is located between residues 14-34 at the N-terminus (Park et al 1999, J. Biol. Chem. 274; 2845).
In an embodiment, the fragment to be incorporated into the immunogenic composition of the invention is the surface exposed fragment containing the elastin binding region (amino acids 1-205). Optionally the fragments do not contain the entire exposed loop but should contain the elastin binding region (amino acids 14-34). An alternative fragment which could be used consists of amino acids forming the second surface exposed loop (amino acids 343-486). Alternative fragments containing up to 1, 2, 5, 10, 20, 50 amino acids less at one or both ends are also possible.
Laminin Receptors
The laminin receptor of S. aureus plays an important role in pathogenicity. A characteristic feature of infection is bloodstream invasion which allows widespread metastatic abscess formation. Bloodstream invasion requires the ability to extravasate across the vascular basement membrane. This is achieved through binding to laminin through the laminin receptor (Lopes et al Science 1985, 229; 275).
Laminin receptors are surface exposed and are present in many strains of staphylococci including S. aureus and S. epidermidis. 
SBI
Sbi is a second IgG binding protein in addition to protein A and it is expressed in most strains of S. aureus (Zhang et al 1998, Microbiology 144; 985).
The N-terminus of the sequence of Sbi has a typical signal sequence with a cleavage site after amino acid 29. Therefore a fragment of Sbi which could be used in an immunogenic composition of the invention starts at amino acid residue 30, 31, 32 or 33 and continues to the C-terminus of Sbi, for example of SEQ ID NO: 26.
The IgG binding domain of Sbi has been identified as a region towards the N-terminus of the protein from amino acids 41-92. This domain is homologous to the IgG binding domains of protein A.
The minimal IgG binding domain of Sbi contains the following sequence (SEQ ID NO:89):—







QTTQNNYVTDQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQEVFSES


          ** ***  *        ***  *  *   *        *


 


LK


 *


 


*denotes amino acids which are similar between IgG


binding domains






In an embodiment, a fragment of Sbi to be included in the immunogenic composition of the invention contains an IgG binding domain. This fragment contains the consensus sequence for an IgG binding domain as designated by * as shown in the above sequence. Optionally the fragment contains or consists of the complete sequence shown above. Optionally, the fragment contains or consists of amino acids 30-92, 33-92, 30-94, 33-94, 30-146, 33-146, 30-150, 33-150, 30-160, 33-160, 33-170, 33-180, 33-190, 33-200, 33-205 or 33-210 of Sbi, for example of SEQ ID NO:26.
A fragment may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions from the sequences indicated.
A fragments may contain multiple repeats (2, 3, 4, 5, 6, 7, 8, 9 or 10) of the IgG binding domain.
EFB-FIB
Fib is a 19 kDa fibrinogen binding protein which is secreted into the extracellular medium by S. aureus. It is produced by all S. aureus isolates tested (Wastfelt and Flock 1995, J. Clin. Microbiol. 33; 2347).
S. aureus clumps in the presence of fibrinogen and binds to fibrinogen coated surfaces. This ability facilitates staphylococcal colonisation of catheters and endothelial cells.
Fib contains a signal sequence at the N-terminus of the protein with a putative cleavage site at about amino acid 30. In an embodiment, the immunogenic composition of the invention comprises or consists of the sequence of the mature protein (from about amino acid 30 to the C-terminus of the protein).
Fbe-EfB/FIG
Fbe is a fibrinogen binding protein that is found in many isolates of S. epidermidis and has a deduced molecular weight of 119 kDa (Nilsson et al 1998. Infect. Immun. 66; 2666). Its sequence is related to that of clumping factor from S. aureus (ClfA). Antibodies against Fbe can block the binding of S. epidermidis to fibrinogen coated plates and to catheters (Pei and Flock 2001, J. Infect. Dis. 184; 52).
Fbe has a putative signal sequence with a cleavage site between amino acids 51 and 52. Therefore a preferred fragment of Fbe contains the mature form of Fbe extending from amino acid 52 to the C-terminus (amino acid 1,092).
The domain of Fbe from amino acid 52 to amino acid 825 is responsible for fibrinogen binding. In an embodiment, the fragment of Fbe consists of or contains amino acids 52-825.
The region between amino acid 373 and 516 of Fbe shows the most conservation between Fbe and ClfA. In an embodiment, the fragment contains amino acids 373-516 of Fbe.
Amino acids 825-1041 of Fbe contains a highly repetitive region composed of tandemly repeated aspartic acid and serine residues.
IsaA/PisA
IsaA is a 29 kDa protein, also known as PisA has been shown to be a immunodominant staphylococcal protein during sepsis in hospital patients (Lorenz et al 2000, FEMS Immunol. Med. Microb. 29; 145).
The first 29 amino acids of the IsaA sequence are thought to be a signal sequence. In an embodiment, the fragment of IsaA to be included in an immunogenic composition of the invention contains amino acid residues 30 onwards, to the end of the coded sequence.
Fibronectin Binding Protein
Fibronectin binding protein A contains several domains that are involved in binding to fibronectin (WO 94/18327). These are called D1, D2, D3 and D4. In an embodiment fragments of fibronectin binding protein A or B comprise or consist of D1, D2, D3, D4, D1-D2, D2-D3, D3-D4, D1-D3, D2-D4 or D1-D4.
Fibronectin binding protein contains a 36 amino acid signal sequence. For example:
VKNNLRYGIRKHKLGAASVFLGTMIVVGMGQDKEAA (SEQ ID NO: 90)
Optionally, the mature protein omitting this signal sequence is included in the immunogenic composition of the invention.
Transporter Proteins
The cell wall of Gram positive bacteria acts as a barrier preventing free diffusion of metabolites into the bacterium. A family of proteins orchestrates the passage of essential nutrients into the bacterium and are therefore essential for the viability of the bacterium. The term transporter protein covers proteins involved in the initial step of binding to metabolites such as iron as well as those involved in actually transporting the metabolite into the bacterium.
Molecular iron is an essential co-factor for bacterial growth. Siderophores are secreted that bind free iron and then are captured by bacterial surface receptors that deliver iron for transport across the cytoplasmic membrane. Iron acquisition is critical for the establishment of human infections so that the generation of an immune response against this class of proteins leads to a loss of staphylococcal viability.
Examples of transporter proteins include Immunodominant ABC transporter (Burnie et al 2000 Infect. Imun. 68; 3200), IsdA (Mazmanian et al 2002 PNAS 99; 2293), IsdB (Mazmanian et al 2002 PNAS 99; 2293, WO 05/09378), IsdC (WO 06/59247), -IsdH/HarA (Pilpa et al 2006, J. Mol. Biol. 360; 435; WO 05/09379) Mg2+ transporter, SitC (Wiltshire and Foster 2001 Infect. Immun. 69; 5198) and Ni ABC transporter.
Immunodominant ABC Transporter
Immunodominant ABC transporter is a well conserved protein which may be capable of generating an immune response that is cross-protective against different staphylococcal strains (Mei et al 1997, Mol. Microbiol. 26; 399). Antibodies against this protein have been found in patients with septicaemia (Burnie et al 2000, Infect. Immun. 68; 3200).
Optional fragments of immunodominant ABC transporter will include the peptides DRHFLN (SEQ ID NO:91), GNYD (SEQ ID NO:92), RRYPF (SEQ ID NO:93), KTTLLK (SEQ ID NO:94), GVTTSLS (SEQ ID NO:95), VDWLR (SEQ ID NO 96), RGFL (SEQ ID NO:97), more preferably KIKVYVGNYDFWYQS (SEQ ID NO:98), TVIWSHDRHFLYNNV (SEQ ID NO:99) and/or TETFLRGFLGRMLFS (SEQ ID NO:100) since these sequences contain epitopes that are recognised by the human immune system.
IsdA-IsdB
The isd genes (iron-regulated surface determinant) of S. aureus encode proteins responsible for haemoglobin binding and passage of haem iron to the cytoplasm, where it acts as an essential nutrient. IsdA and IsdB are located in the cell wall of staphylococci. IsdA appear to be exposed on the surface of bacterium since it is susceptible to proteinase K digestion. IsdB was partially digested suggesting that it is partially exposed on the surface of the bacterium (Mazmanian et al 2003 Science 299; 906).
IsdA and IsdB are both 29 kDa proteins which bind heme. Their expression is regulated by the availability of iron via the Fur repressor. Their expression will be high during infection in a host where the concentration of iron will be low.
They are also known as FrpA and FrpB (Morrissey et al 2002, Infect. Immun. 70; 2399). FrpA and FrpB are major surface proteins with a high charge. They have been shown to provide a major contribution to adhesion to plastic.
In an embodiment, the immunogenic composition of the invention comprises a fragment of IsdA and/or IsdB which is described in WO 01/98499 or WO 03/11899.
HarA
HarA is a further iron-regulated protein. It contains a signal peptide of amino acids 1-40. Optionally, the HarA present in the immunogenic compositions of the invention omits the signal peptide.
HarA contains three NEAT domains from amino acid 101-232, from amino acid 341-471 and from amino acid 539-664. For example, a fragment of HarA comprises or consists of amino acids 101-232, 101-471, 101-664, 341-471. 341-664 or 539-664, optionally from the sequence of SEQ ID NO 69.
HarA contains a Gram plus anchor domain: from aa 853 to aa 892. Optionally, a fragment of HarA omits this domain.
Signal peptide length: 40 amino acids—underlined in first row of sequence
NEAT domains—three underlined internal regions.
Gram+anchor domain—underlined region on bottom line of sequence.




Toxins and Regulators of Virulence
Members of this family of proteins include toxin such as alpha toxin, hemolysin, enterotoxin B, Panton Valentine Leucocidin (VPL) (Morinaga et al Microbiol. Immunol. 47; 81-90, 2003) and TSST-1 as well as proteins that regulate the production of toxins such as RAP.
Alpha Toxin (Hla)
Alpha toxin is an important virulence determinant produced by most strains of S. aureus. It is a pore forming toxin with haemolytic activity. Antibodies against alpha toxin have been shown to neutralise the detrimental 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 susceptible to the effects of alpha toxin.
The high toxicity of alpha toxin requires that it should be detoxified before being used as an immunogen. This can be achieved by chemical treatment, for instance by treating with formaldehyde, glutaraldehyde of other cross-linking reagents or by chemically conjugating it to bacterial polysaccharides as described below.
A further way of removing toxicity is to introduce point mutations that remove toxicity while retaining the antigenicity of the toxin. The introduction of a point mutation at amino acid 35 of alpha toxin where a histidine residue is replaced with a leucine residue results in the removal of toxicity whilst retaining immunogenicity (Menzies and Kernodle 1996; Infect. Immun. 64; 1839). Histidine 35 appears to be critical for the proper oligomerization required for pore formation and mutation of this residue leads to loss of toxicity.
When incorporated into immunogenic compositions of the invention, alpha toxin is optionally detoxified by mutation of His 35, for example by replacing His 35 with Leu or Arg. In an alternative embodiment, alpha toxin is detoxified by conjugation to other components of the immunogenic composition, for example capsular polysaccharides or PNAG, most preferably to S. aureus type 5 polysaccharide and/or S. aureus Type 8 polysaccharide and/or PNAG.
RNA III Activating Protein (RAP)
RAP is not itself a toxin, but is a regulator of the expression of virulence factors. RAP is produced and secreted by staphylococci. It activates the agr regulatory system of other staphylococci and activates the expression and subsequent release of virulence factors such as hemolysin, enterotoxin B and TSST-1.
Other Immunodominant Proteins
Accumulation-Associated Protein (Aap)
Aap is a 140 kDa protein which is essential for the accumulation of S. epidermidis strains on surfaces (Hussain et al Infect. Immun. 1997, 65; 519). Strains expressing this protein produced significantly larger amounts of biofilm and Aap appear to be involved in biofilm formation. Antibodies against Aap are able to inhibit biofilm formation and inhibit the accumulation of S. epidermidis. Sequences which could be added to a vaccine are disclosed in WO 05/86663.
Staphylococcal Secretory Antigen SsaA
SsaA is a strongly immunogenic protein of 30 kDa found in both S. aureus and S. epidermidis (Lang et al 2000 FEMS Immunol. Med. Microbiol. 29; 213). Its expression during endocarditis suggested a virulence role specific to the pathogenesis of the infectious disease.
SsaA contains an N-terminal leader sequence and a signal peptidase cleavage site. The leader peptide is followed by a hydrophilic region of approximately 100 amino acids from residue 30 to residue 130.
An optional fragment of SsaA to be incorporated into the immunogenic composition of the invention is made up of the mature protein (amino acids 27 to the C-terminus or amino acids 30 to the C-terminus).
A further optional fragments contains the hydrophilic area of SsaA from amino acid 30 to amino acid 130. Further optional sequences and fragments are disclosed in WO 05/115113,
Penicillin Binding Protein 4
Penicillin binding protein 4 is described in Henze et al Antimicrobial Agents and Chemotherapy 38: 2415, 1995 and WO 06/33918.
Preferred Combinations
Staphylococcal infections progress through several different stages. For example, the staphylococcal life cycle involves commensal colonisation, initiation of infection by accessing adjoining tissues or the bloodstream, anaerobic multiplication in the blood, interplay between S. aureus virulence determinants and the host defense mechanisms and induction of complications including endocarditis, metastatic abscess formation and sepsis syndrome. Different molecules on the surface of the bacterium will be involved in different steps of the infection cycle. By targeting the immune response against a combination of particular antigens involved in different processes of Staphylococcal infection, multiple aspects of staphylococcal function are affected and this can result in good vaccine efficacy.
In particular, combinations of certain antigens from different classes, some of which are involved in adhesion to host cells, some of which are involved in iron acquisition or other transporter functions, some of which are toxins or regulators of virulence and immunodominant antigens can elicit an immune response which protects against multiple stages of infection.
Some combinations of antigens are particularly effective at inducing an immune response. This can be measured either in animal model assays as described in the examples and/or using an opsonophagocytic assay as described in the examples. Without wishing to be bound by theory, such effective combinations of antigens are thought to be enabled by a number of characteristics of the immune response to the antigen combination. The antigens themselves are usually exposed on the surface of Staphylococcal cells, they tend to be conserved but also tend not to be present in sufficient quantity on the surface cell for an optimal bactericidal response to take place using antibodies elicited against the single antigen. Combining the antigens of the invention can result in a formulation eliciting an advantageous combination of antibodies which interact with the Staphylococcal cell beyond a critical threshold. At this critical level, sufficient antibodies of sufficient quality bind to the surface of the bacterium to allow either efficient killing by complement or neutralisation of the bacterium. This can be measured in either an animal challenge model or an opsonisation assay as described in the examples.
In an embodiment, processes of the invention mix a plurality of proteins selected from at least two different categories of protein, having different functions within Staphylococci. Examples of such categories of proteins are extracellular binding proteins, transporter proteins such as Fe acquisition proteins, toxins or regulators of virulence and other immunodominant proteins, thus making an immunogenic composition of the invention.
In an embodiment, the process or immunogenic composition of the invention further use/comprises a number of proteins equal to or greater than 2, 3, 4, 5 or 6 selected from 2 or 3 different groups selected from;

    
    
        Group a) extracellular component binding proteins;
        Group b) transporter proteins;
        Group c) toxins or regulators of virulence.
    
    


In an embodiment, the immunogenic composition of the invention further comprises a number of proteins equal to or greater than 2, 3, 4, 5 or 6 selected from 2 or 3 of the following groups:

    
    
        group a)—at least one staphylococcal extracellular component binding protein or fragment thereof selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, Protein A, EbhA, EbhB, Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdeD, SdrE, SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, SasH, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein, coagulase, Fig and MAP;
        group b)—at least one staphylococcal transporter protein or fragment thereof selected from the group consisting of Immunodominant ABC transporter, IsdA, IsdB, IsdH/HarA Mg2+ transporter, SitC and Ni ABC transporter;
        group c)—at least one staphylococcal regulator of virulence, toxin or fragment thereof selected from the group consisting of alpha toxin (Hla), alpha toxin H35R mutant, RNA III activating protein (RAP);
        group d)—at least one staphylococcal structural protein or immunogenic fragment thereof selected from the group consisting of MRPII and autolysin.
    
    


These particular immunogenic composition may include at least one staphylococcal saccharide and/or protein as part of the saccharide-protein conjugate of the invention and at least one staphylococcal antigen which is not part of the saccharide-protein conjugate of the invention in order to complete the combination.
In an embodiment, the immunogenic composition of the invention contains at least one protein selected from group a) and an additional protein selected from group b) and/or group c).
In a further embodiment, the immunogenic composition of the invention contains at least one antigen selected from group b) and an additional protein selected from group c) and/or group a).
In a further embodiment, the immunogenic composition of the invention contains at least one antigen selected from group c) and an additional protein selected from group a) and/or group b).
In a further embodiment, the process of the invention involves the addition of any of the staphylococcal proteins listed in WO 02/59148, WO 05/09378, WO 05/09379, WO 05/86663, WO 05/115113, WO 06/33918, WO 06/78680, WO 06/121664, WO 07/01361, WO 02/94868, U.S. Pat. No. 6,380,370, WO 04/87746, WO 01/98499 or WO 03/11899.
In an embodiment, the immunogenic composition of the invention comprises a dose of each saccharide conjugate between 0.1 and 20 μg, 2 and 10 μg, 2 and 6 μg or 4 and 7 μg of saccharide per administered dose. In an embodiment, the method of the invention mixes between 0.1 and 20 μg, 2 and 10 μg, 2 and 6 μg or 4 and 7 μg of each saccharide.
“Around” or “approximately” are defined as within 10% more or less of the given figure for the purposes of the invention.
In an embodiment, the immunogenic composition of the invention is adjusted to or buffered at, or adjusted to between pH 7.0 and 8.0, pH 7.2 and 7.6 or around or exactly pH 7.4.
The immunogenic composition or vaccines of the invention are optionally lyophilised in the presence of a stabilising agent for example a polyol such as sucrose or trehalose.
Optionally, the immunogenic composition or vaccine of the invention contains an amount of an adjuvant sufficient to enhance the immune response to the immunogen. Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875. Equally, the method of the invention optionally comprises a step of adding at least one of the above adjuvants.
As with all immunogenic compositions or vaccines, the immunologically effective amounts of the immunogens must be determined empirically. Factors to be considered include the immunogenicity, whether or not the immunogen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, route of administrations and the number of immunising dosages to be administered.
The active agent can be present in varying concentrations in the pharmaceutical composition or vaccine of the invention. Typically, the minimum concentration of the substance is an amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or homogeneously suspended within the initial mixture. For instance, the minimum amount of a therapeutic agent is optionally one which will provide a single therapeutically effective dosage. For bioactive substances, the minimum concentration is an amount necessary for bioactivity upon reconstitution and the maximum concentration is at the point at which a homogeneous suspension cannot be maintained. In the case of single-dosed units, the amount is that of a single therapeutic application. Generally, it is expected that each dose will comprise 1-100 μg of protein antigen, optionally 5-50 μg or 5-25 μg. For example, doses of bacterial saccharides are 10-20 μg, 5-10 μg, 2.5-5 μg or 1-2.5 μgof saccharide in the conjugate.
The vaccine preparations of the present invention may be used to protect or treat a mammal (for example a human patient) susceptible to infection, by means of administering said vaccine via systemic or mucosal route. A human patient is optionally an infant (under 12 months), a toddler (12-24, 12-16 or 12-14 months), a child (2-10, 3-8 or 3-5 years) an adolescent (12-21, 14-20 or 15-19 years) or an adult. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage). Although the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance if saccharides are present in a vaccine these could be administered separately at the same time or 1-2 weeks after the administration of a bacterial protein vaccine for optimal coordination of the immune responses with respect to each other). In addition to a single route of administration, 2 different routes of administration may be used. For example, viral antigens may be administered ID (intradermal), whilst bacterial proteins may be administered IM (intramuscular) or IN (intranasal). If saccharides are present, they may be administered IM (or ID) and bacterial proteins may be administered IN (or ID). In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses.
Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
In a further aspect of the invention there is provided an immunogenic composition comprising a saccharide-protein carrier conjugate and a staphylococcal antigen obtainable or obtained by the method of the invention.
A use of the immunogenic composition or vaccine of the invention in the manufacture of a medicament for the prevention or treatment of disease, and a method of preventing or treating disease comprising the step of administering an effective dose of the immunogenic composition or vaccine of the invention to a patient in need thereof is further provided. The use or method may be such that the disease is caused by a bacterium selected from a list consisting of: N. meningitidis, Streptococcus pneumoniae, M. catarrhalis, Group B Streptococcus, Staphylococcus aureus, Salmonella typhi, Vibrio cholerae, E. coli, and H. influenzae. 
The terms “comprising”, “comprise” and “comprises” herein are intended by the inventors to be optionally substitutable with the terms “consisting of”, “consist of” and “consists of”, respectively, in every instance.
All references or patent applications cited within this patent specification are incorporated by reference herein.
The invention is illustrated in the accompanying examples. The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
EXAMPLES
Example 1
Preparation of Polysaccharide Conjugates
S. aureus Capsular Polysaccharide Type 8-TT Conjugate:
PS Derivatization
Activation and coupling were performed at room temperature under continuous stirring. 30 mg of native polysaccharide were diluted to obtain a final polysaccharide concentration of 5 mg/ml in water. The solution was adjusted to pH 5.0 with 0.5N HCl and then 66 μg of ADH were added (2.2 mg/mg PS). After complete dissolution, 60 mg of EDAC were added (2 mg/mg PS). After 70 min the pH was raised to pH 7.5 with 1N NaOH to stop the reaction. Free ADH was removed by purification on SEPHACRYL® S100HR (XK 16/40). The flow-rate was fixed at 60 ml/h using 0.2 M NaCl as elution buffer. A size reduction was done by sonication of 15 min allowing a sterile filtration on millex filter (0.22 μm).
Coupling
Tetanus toxoid was added to 5 to 10 mg of derivatized polysaccharide in 0.2M NaCl and the pH was adjusted to pH 5.0 or pH 6.0 by addition of 0.5N HCl. EDAC was dissolved in 0.1M Tris buffer pH 7.5 and then added over a period of 10 min (1⁄5 vol each 2 min). According to the conditions used (see Table 6), the reaction was stopped after between 30 and 180 minutes by addition of 1M Tris-HCl pH 7.5. Prior to purification on SEPHACRYL® S400HR, the conjugate was clarified using a 5 μm Minisart filter. Alternatively, the conjugate was clarified by a 5 minute sonication step. The conjugate was then injected on SEPHACRYL® S400HR (XK50/100). The flow-rate was fixed at 30 ml/h using 150 mM NaCl as elution buffer. The elution pool was selected on the basis of resorcinol and μBCA profiles (which measure polysaccharide and protein dosage respectively). The conjugate was filtered on a 0.22 μm sterilizing membrane (Millipack 20) at 10 ml/min.











TABLE 6






Coupling
[PS (AH)]
[TT (AH)]
[reagent EDAC]


Conjugate
time
(mg/ml)
(mg/ml)
(mg/mg PS)



















SA08-TT011
40 min
5
10
 0.5/1


SA08-TT015*
180 min 
2.5
5.0
0.25/1


SA08-TT017
30 min
3.75
7.5
0.25/1


SA08-TT018
50 min
3.75
7.5
0.10/1





*coupling done at pH 6.0






The resulting conjugates have the following characteristics shown in Table 7:











TABLE 7






In. TT/PS
F. TT/PS
Y. PS
Filtr.


Conjugate
ratio(w/w)
ratio(w/w)
rec (%)
Yield (%)



















SA08-TT011
2/1
2.43/1
48
99


SA08-TT015
2/1
2.40/1
53
104


SA08-TT017
2/1
2.41/1
44
107


SA08-TT018
2/1
2.40/1
42
106









S. aureus polysaccharide type 8 was also treated by microfluidization before derivatization with ADH
PS Derivatization
Activation and coupling are performed at room temperature under continuous stirring. 200 mg of sized polysaccharide are diluted to obtain a final PS concentration of 10 mg/ml in water. Then 440 mg of ADH were added (2.2 mg/mg PS). The solution was adjusted to pH 4.7 with 1N HCl before the addition of 400 mg of EDAC (2 mg/mg PS). After 60 min the pH was raised to pH 7.5 with 5M NaOH to stop the reaction. The mixture was concentrated on Amicon Ultra (cut-off 10.000 MWCO). Prior to purification on SEPHACRYL® S200HR (XK16/100), the conjugate was clarified using a 5 μm Minisart filter. The flow-rate was fixed at 30 ml/h using 0.150 M NaCl as elution buffer.
Coupling
100 mg of TT was added to 50 mg of derivatized polysaccharide in 0.15M NaCl. The pH was adjusted to pH 5.0±0.02 by addition of 0.3N HCl. EDAC was dissolved in 0.1M Tris buffer pH 7.5 and then added over a period of 10 min ( 1/10 vol each minute). According to the conditions used (see Table 8), the reaction was stopped after between 30 and 180 minutes by addition of 1M Tris-HCl pH 7.5. Prior to purification on SEPHACRYL® S400HR, the conjugate was clarified using a 5 μm Minisart filter. The conjugate was then injected on SEPHACRYL® S400HR (XK50/100). The flow-rate was fixed at 60 ml/h using 150 mM NaCl as elution buffer. The elution pool was selected on the basis of resorcinol and μBCA profiles (which measure polysaccharide and protein dosage respectively). Then, the conjugate was filtered on a 0.22 μm sterilizing membrane (Millipack 20) at 10 ml/min.











TABLE 8






Coupling
[PS-AH]
[TT]
[EDAC]


Conjugate
time
(mg/ml)
(mg/ml)
(mg/mg PS)



















SA08-TT045
65 min
3.75
7.5
0.1


SA08-TT046
45 min
3.75
7.5
0.2


SA08-TT047
30 min
5.0
15.0
0.2


SA08-TT048
120 min 
5.0
10.0
0.05


SA08-TT049*
50 min
5.0
10.0
0.1





*EDAC added in “one time”

















TABLE 9






In. TT/PS
F. TT/PS
Y. PS
Filtr.


Conjugate
ratio(w/w)
ratio(w/w)
rec (%)
Yield (%)







SA08-TT045
2/1
2.20/1
57
101


SA08-TT046
2/1
2.80/1


SA08-TT047
3/1
Gel- Not purified
—
—


SA08-TT048
2/1
3.35
30
101


SA08-TT049
2/1
3.5 
24
106









Example 1a
Preparation of Meningococcal MenA and MenC Capsular Polysaccharide Conjugate According to the Invention
MenC-TT conjugates were produced using native polysaccharides (of over 150 kDa as measured by MALLS) or were slightly microfluidised. MenA-TT conjugates were produced using either native polysaccharide or slightly microfluidised polysaccharide of over 60 kDa as measured by the MALLS method of example 2. Sizing was by microfluidisation using a homogenizer EMULSIFLEX® C-50 apparatus. The polysaccharides were then filtered through a 0.2 μm filter.
In order to conjugate MenA capsular polysaccharide to tetanus toxoid via a spacer, the following method was used. The covalent binding of the polysaccharide and the spacer (ADH) is carried out by a coupling chemistry by which the polysaccharide is activated under controlled conditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). The spacer reacts with the cyanylated PS through its hydrazino groups, to form a stable isourea link between the spacer and the polysaccharide.
A 10 mg/ml solution of MenA (pH 6.0) [3.5 g] was treated with a freshly prepared 100 mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v)) to obtain a CDAP/MenA ratio of 0.75 (w/w). After 1.5 minutes, the pH was raised to pH 10.0. Three minutes later, ADH was added to obtain an ADH/MenA ratio of 8.9. The pH of the solution was decreased to 8.75 and the reaction proceeded for 2 hours maintaining this pH (with temperature kept at 25° C.).
The PSAAH solution was concentrated to a quarter of its initial volume and then diafiltered with 30 volumes of 0.2M NaCl using a Filtron Omega membrane with a cut-off of 10 kDa, and the retentate was filtered.
Prior to the conjugation (carbodiimide condensation) reaction, the purified TT solution and the PSAAH solution were diluted to reach a concentration of 10 mg/ml for PSAAH and 10 mg/ml for TT.
EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was added to the PSAH solution (2 g saccharide) in order to reach a final ratio of 0.9 mg EDAC/mg PSAAH. The pH was adjusted to 5.0. The purified tetanus toxoid was added with a peristaltic pump (in 60 minutes) to reach 2 mg TT/mg PSAAH. The resulting solution was left 60 min at +25° C. under stirring to obtain a final coupling time of 120 min. The solution was neutralised by addition of 1M Tris-Hcl pH 7.5 ( 1/10 of the final volume) and left 30 minutes at +25° C. then overnight at +2° C. to +8° C.
The conjugate was clarified using a 10 μm filter and was purified using a SEPHACRYL® S400HR column (Pharmacia, Sweden). The column was equilibrated in 10 mM Tris-HCl (pH 7.0), 0.075 M NaCl and the conjugate (approx. 660 mL) was loaded on the column (+2° C. to +8° C.). The elution pool was selected as a function of optical density at 280 nm. Collection started when absorbance increased to 0.05. Harvest continued until the Kd reached 0.30. The conjugate was filter sterilised at +20° C., then stored at +2° C. to +8° C. The resultant conjugate had a polysaccharide:protein ratio of 1:2-1:4 (w/w).
In order to conjugate MenC capsular polysaccharide to tetanus toxoid via a spacer, the following method was used. The covalent binding of the polysaccharide and the spacer (ADH) is carried out by a coupling chemistry by which the polysaccharide is activated under controlled conditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). The spacer reacts with the cyanylated PS through its hydrazino groups, to form a stable isourea link between the spacer and the polysaccharide.
A 20 mg/ml solution of MenC (pH6.0) (3.5 g) was treated with a freshly prepared 100 mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v)) to obtain a CDAP/MenC ratio of 1.5 (w/w). After 1.5 minutes, the pH was raised to pH 10.0. At activation pH 5M NaCl was added to achieve a final concentration of 2M NaCl. Three minutes later, ADH was added to obtain an ADH/MenC ratio of 8.9. The pH of the solution was decreased to 8.75 and the reaction proceeded for 2 hours (retained at 25° C.).
The PSCAH solution was concentrated to a minimum of 150 mL and then diafiltered with 30 volumes of 0.2M NaCl using a Filtron Omega membrane with a cut-off of 10 kDa, and the retentate was filtered.
Prior to the conjugation reaction, the purified TT solution and the PSCAH solution (2 g scale) were diluted in 0.2M NaCl to reach a concentration of 15 mg/ml for PSCAH and 20 mg/ml for TT.
The purified tetanus toxoid was added to the PSCAH solution in order to reach 2 mg TT/mg PSCAH. The pH was adjusted to 5.0. EDAC (16.7 mg/ml in Tris 0.1M pH 7.5) was added with a peristaltic pump (in 10 minutes) to reach a final ratio of 0.5 mg EDAC/mg PSCAH. The resulting solution was left 110 min at +25° C. under stirring and pH regulation to obtain a final coupling time of 120 min. The solution was then neutralized by addition of 1M Tris-Hcl pH 9.0 ( 1/10 of final volume) and left 30 minutes at +25° C. then overnight at +2° C. to +8° C.
The conjugate was clarified using a 10 μm filter and was purified using a SEPHACRYL® S400HR column (Pharmacia, Sweden). The column was equilibrated in 10 mM Tris-HCl (pH 7.0), 0.075 M NaCl and the conjugate (approx. 460 mL) was loaded on the column (+2° C. to +8° C.). The elution pool was selected as a function of optical density at 280 nm. Collection started when absorbance increased to 0.05. Harvest continued until the Kd reached 0.20. The conjugate was filter sterilised at +20° C., then stored at +2° C. to +8° C. The resultant conjugate had a polysaccharide:protein ratio of 1:2-1:4 (w/w).
Various experiments adding EDAC over 10-45 minutes were carried out—in each case good quality MenC conjugates resulted. If, however the TT carrier was added last slowly to the MenC-ADH+EDAC mix this led to a gel—a conjugate that could not be purified.
Experiments were also carried out adding the EDAC all at once into the reaction but the final TT/PS ratio (2.7/1) (w/w) of the conjugate was lower than for the conjugate obtained via the reaction where EDAC was added over 10 minutes (3.3/1); furthermore the αTT and aPS antigenicity were both lower than that measured in respect of the conjugate made by the reaction where EDAC was added over 10 minutes.
Note on Approximate % Derivatisation of the Polysaccharides
MenCAH: after CDAP treatment with ADH about 3.47% of hydroxyl groups were derivatized with ADH (with an estimation of two available hydroxyl groups per repeat subunit). For MenA: about 11.5% of hydroxyl groups derivatized with ADH (considering there is only one available hydroxyl group per repeat unit).
Example 2
Determination of Molecular Weight Using MALLS
Detectors were coupled to a HPLC size exclusion column from which the samples were eluted. On one hand, the laser light scattering detector measured the light intensities scattered at 16 angles by the macromolecular solution and on the other hand, an interferometric refractometer placed on-line allowed the determination of the quantity of sample eluted. From these intensities, the size and shape of the macromolecules in solution can be determined.
The mean molecular weight in weight (Mw) is defined as the sum of the weights of all the species multiplied by their respective molecular weight and divided by the sum of weights of all the species.

    
    
        a) Weight-average molecular weight: -Mw-
    
    






  
    M
    w
  
  =
  
    
      
        ∑
        
          
            W
            i
          
          ·
          
            M
            i
          
        
      
      
        ∑
        
          W
          i
        
      
    
    =
    
      
        m
        2
      
      
        m
        1
      
    
  




    
    
        b) Number-average molecular weight: -Mn-
    
    






  
    M
    n
  
  =
  
    
      
        ∑
        
          
            N
            i
          
          ·
          
            M
            i
          
        
      
      
        ∑
        
          N
          i
        
      
    
    =
    
      
        m
        1
      
      
        m
        0
      
    
  




    
    
        c) Root mean square radius: -Rw- and R2w is the square radius defined by:
    
    






  
    
      R
      2
    
    ⁢
    w
    ⁢
    
      
    
    ⁢
    or
    ⁢
    
      
    
    ⁢
    
      (
      
        r
        2
      
      )
    
    ⁢
    w
  
  =
  
    
      ∑
      
        
          m
          i
        
        ·
        
          r
          i
          2
        
      
    
    
      ∑
      
        m
        i
      
    
  




    
    
        
        
            (-mi- is the mass of a scattering centre i and -ri- is the distance between the
            scattering centre i and the center of gravity of the macromolecule).
        
        
        d) The polydispersity is defined as the ratio -Mw/Mn-.
    
    


Meningococcal polysaccharides were analysed by MALLS by loading onto two HPLC columns (TSKG6000 and 5000PWxl) used in combination. 250 of the polysaccharide were loaded onto the column and was eluted with 0.75 ml of filtered water. The polyaccharides are detected using a light scattering detector (Wyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and an inferometric refractometer (Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498 nm).
The molecular weight polydispersities and recoveries of all samples were calculated by the Debye method using a polynomial fit order of 1 in the Astra 4.72 software.
Example 3
Immunogenicity of S. aureus PS8-TT and dPNAG-TT Conjugates
Groups of 30 mice were inoculated subcutaneously with S. aureus PS8-TT conjugate at a saccharide dose of 3 μg, either unadjuvanted or combined with adjuvant A, on days 0, 14, 28 and 42. On day 0, the mice received a first saccharide dose including between 0.001 and 0.013 μg. The further three immunisations were done with a dose of 0.3 μg in saline. On day 55 serum was collected from the mice and each serum sample was tested by ELISA to assess the immune response against PS8. Groups of 10 mice were used in the control groups and these were inoculated with either saline or saline containing adjuvant A.
The purified PS8 was coated at 2 μg/ml in phosphate buffered saline (PBS) on high binding microtitre plates (Nunc Maxisorp) overnight at 4° C. The plates were blocked with PBS-BSA 1% for 30 min at room temperature with agitation. The mice antisera were prediluted 1/100, then further twofold dilutions were made in microplates which were incubated at 37° C. for 1 hour. After washing, bound murine antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated affiniPure Goat Anti-Mouse IgG (H+L) (ref: 115-035-003) diluted 1:5000 in PBS-TWEEN® 0.05%. The detection antibodies were incubated for 30 minutes at room temperature with agitation. The color was developed using 4 mg OPD (Sigma)+5 μl H2O2 per 10 ml pH 4.5 0.1M citrate buffer for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μl HCl, and the optical density was read at 490 nm relative to 650 nm.
The results were expressed in mid-point titers and the GMT was calculated for the 30 samples (10 for controls). The results are shown in Table 14 below.











TABLE 14








Anti-PS8 titre (GMT)
Anti-PS8 titre (GMT)



Conjugate
nonadsorbed
Adjuvant A




















SA08-TT011
4714
2109



SA08-TT015
2806
5631



SA08-TT017
3770
4396



SA08-TT018
5349
4748



Control
50
50










Groups of 30 mice were inoculated subcutaneously with S. aureus dPNAG-TT conjugates (containing dPNAG which was between 10% and 30% N-acetylated) at a saccharide dose of 0.3 μg in 200 mM NaCl, either unadjuvanted or combined with adjuvant A. The mice received three inoculations on days 0, 14 and 28. On day 41 or 42 serum was collected from the mice and each serum sample was tested by ELISA to assess the immune response against PNAG. Groups of 10 mice were used in the control groups and these were inoculated with saline or with adjuvant alone.

## Claims

1. A method of making an immunogenic composition comprising a conjugation step of conjugating a saccharide to a protein carrier to make a saccharide-protein conjugate using carbodiimide condensation chemistry, wherein the saccharide comprises, or has been derivatised to comprise, amino and/or carboxyl groups, and wherein the protein carrier comprises, or has been derivatised to comprise, amino and/or carboxyl groups, comprising the steps of:
I) if the protein carrier comprises both amino and carboxyl groups and the saccharide comprises either amino or carboxyl groups:
a) mixing the saccharide and aliquot of carbodiimide required to perform the conjugation, and
b) adding aliquot of the protein carrier required over a time period of 35 seconds to 6 hours to form a saccharide-protein conjugate;
II) if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises either amino or carboxyl groups:
a) mixing the protein carrier and aliquot of carbodiimide required to perform the conjugation, and
b) adding aliquot of saccharide required over a time period of 10 minutes to 4 hours to form a saccharide-protein conjugate; and
III) if the saccharide comprises both amino and carboxyl groups and the protein carrier comprises both amino and carboxyl groups:
a) mixing the protein carrier and the saccharide, and
b) adding aliquot of carbodiimide required to perform the conjugation over a time period of 35 seconds to 6 hours to form a saccharide-protein conjugate;
and a further step of mixing the saccharide-protein conjugate of I), II), or III) with a staphylococcal antigen.

2. The method of claim 1, wherein the carbodiimide is EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide).

3. The method of claim 1, wherein the saccharide and/or the protein carrier has been derivatised to comprise the amino or the carboxyl groups.

4. The method of claim 1, wherein in step b) the aliquot of the carbodiimide, the aliquot of the saccharide or the aliquot of the protein carrier is added at a constant rate using a pump.

5. The method of claim 1, wherein in step b) the aliquot of carbodiimide, the aliquot of saccharide or the aliquot of protein carrier is added in stages over the time period.

6. The method of claim 5, wherein at least one quarter of the aliquot is added over the first half of the time period, and at least one quarter of the aliquot over the second half of the time period.

7. The method of claim 5, wherein the aliquot is added in 4-100 stages.

8. The method of claim 7, wherein if one of the stages takes place at time zero of the time period, each subsequent stage takes place at a time period which is evenly spaced throughout the time period.