Immunogenic composition comprising a haemophilus influenzae type b saccharide conjugate

IMMUNOGENIC COMPOSITION COMPRISING A HAEMOPHILUS INFLUENZAE

TYPE B SACCHARIDE CONJUGATE

The present application relates to Immunogenic compositions and vaccines comprising a Hib saccharide conjugate and at least two further bacterial saccharide conjugates, processes for making such immunogenic compositions and vaccines, uses and methods of immunisation using the immunogenic composition and vaccine.

Bacterial polysaccharides have been shown to be effective immunogens for use in vaccines, particularly when conjugated to a carrier protein. Commercial conjugate vaccines are available against Haemophilus influenzae type b (Hibtiter® Wyeth-Lederle), pneumococcal polysaccharides (Prevnar® -Wyeth-Lederle) and meningococcal polysaccharides (Meningitec® - Wyeth-Lederle and Menactra®- Sanofi).

Immunogenic compositions and vaccines comprising a Hib conjugate and further bacterial saccharide conjugates have also been described. For instance WO 02/00249 discloses immunogenic compositions comprising a Hib PRP conjugate and further polysaccharide or oligosaccharide conjugates wherein the polysaccharide conjugates are not adsorbed onto adjuvant, particularly aluminium salts. The clinical trial results presented use the same doses of all bacterial polysaccharides.

Choo et al in Pediatr. Infect. Dis.J. (2000) 19; 854-62 describes inoculation of young children with a 7-valent pneumococcal conjugate vaccine mixed with a Haemophilus influenzae type b(hib) conjugate vaccine known as HbOC. The dose of hib conjugate administered was 5 times higher than the dose of each of the pneumococal polysaccharide conjugates administered.

The present invention concerns the provision of a combination vaccine comprising a Hib conjugate and further bacterial saccharide conjugates which is capable of eliciting an improved immunogenic response due to the optimisation of the doses of the Hib conjugate and other bacterial polysaccharide conjugates.

Accordingly, a first aspect of the invention provides an immunogenic composition comprising a Hib saccharide conjugate and at least two further bacterial saccharide conjugates wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of the at least two further bacterial saccharide conjugates.

Detailed description

The immunogenic composition of the invention comprises a Hib saccharide conjugate and at least two further bacterial saccharide conjugates wherein the Hib conjugate is

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PCT/EP2006/006210 present in a lower saccharide dose than the mean saccharide dose of the at least two further bacterial saccharide conjugates. Alternatively, the Hib conjugate is present in a lower saccharide dose than the saccharide dose of each of the at least two further bacterial saccharide conjugates. For example, the dose of the Hib conjugate may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% lower than the mean or lowest saccharide dose of the at least two further bacterial saccharide conjugates.

The term “saccharide” includes polysaccharides or oligosaccharides. Polysaccharides are isolated from bacteria or isolated from bacteria and sized to some degree by known methods (see for example EP497524 and EP497525) and optionally 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 “mean dose’ is determined by adding the doses of all the further polysaccharides and dividing by the number of further polysaccharides. The “dose” is in the amount of immunogenic composition or vaccine that is administered to a human.

Polysaccharides are optionally sized up to 1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 times from the size of the polysaccharide isolated from bacteria.

“Sized by a factor up to x2” means that the polysaccharide is subject to a process intended to reduce the size of the polysaccharide but to retain a size more than half the size of the native polysaccharide. X3, x4 etc. are to be interpreted in the same way i.e. the polysaccharide 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 respectively.

The size of MenA saccharide is for example 5-200kDa, 10-20kDa, 5-10kDa, 20-30kDa, 20-40kDa, 40-80kDa, 60-80kDa, 60-70kDa or 70-80kDa.

The size of MenC saccharide is for example 5-200kDa,10-20kDa, 5-lOkDa, 5-15kDa, 2050kDa, 50-100kDa, 100-150kDa, 150-21 OkDa.

The size of MenW saccharide is for example 5-200kDa, 10-20kDa, 5-1 OkDa, 20-50kDa, 50-100kDa, 100-150kDa or 120-140kDa.

The size of MenY saccharide is for example 5-200kDa, 10-20kDa, 5-1 OkDa, 20-50kDa, 50-100kDa, 100-l40kDa, 140-170kDa or 150-160kDa as determined by MALLS.

In an embodiment, the polydispersity of the saccharides is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or 11.05 and after conjugation to a carrier protein, the polydispersity of the conjugate is 1.02.0.1.0-1.5,1.0-1.2 or 1.5-2.0. All polydispersity measurements are by MALLS.

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For MALLS analysis of meningococcal saccharides, two columns (TSKG6000 and 5000PWxl TOSOH Bioscience) 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 10mW argon laser at 488nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm).

A Hib saccharide is the polyribosyl phosphate (PRP) capsular polysaccharide or oligosaccharide of Haemophilus influenzae type b.

“At least two further bacterial saccharide conjugates” refers to at least two saccharide conjugates in which the saccharides are different from Hib and from each other. The at least two further bacterial saccharide conjugates may be derived from one or more of Neisseria meningitidis. Streptococcus pneumoniae, Group A Streptococci, Group B Streptococci, S. typhi, Staphylococcus aureus or Staphylococcus epidermidis. In an embodiment, the immunogenic composition comprises capsular polysaccharides or oligosaccharides derived from one or more of serogroups A, B, C, W135 and Y of Neisseria meningitidis. A further embodiment comprises capsular polysaccharides or oligosaccharides derived from Streptococcus pneumoniae. The pneumococcal capsular polysaccharide or oligosaccharide antigens are optionally selected from serotypes 1. 2, 3, 4, 5, 6A, 6B, 7F. 8. 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A. 19F, 20, 22F, 23F and 33F (for example from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). A further embodiment comprises the Type 5, Type 8 or 336 capsular polysaccharides or oligosaccharides of Staphylococcus aureus. A further embodiment comprises the Type I, Type II or Type III capsular polysaccharides of Staphylococcus epidermidis. A further embodiment comprises the Vi saccharide (poly or oligosaccharide) from S. typhi. A further embodiment comprises the Type la, Type le. Type II or Type III capsular polysaccharides or oligosaccharides of Group B streptocoocus. A further embodiment comprises the capsular polysaccharides or oligosaccharides of Group A streptococcus, optionally further comprising at least one M protein or multiple types of M protein. In an embodiment, the immunogenic composition of the invention further comprises an antigen from N. meningitidis serogroup B. The antigen is optionally a capsular polysaccharide from N. meningitidis serogroup B (MenB) or a sized polysaccharide or oligosaccharide derived therefrom. The antigen is optionally an outer membrane vesicle preparation from N. meningitidis serogroup B as described in EP301992, WO 01/09350, WO 04/14417, WO 04/14418 and WO 04/14419.

In an embodiment, the at least two further bacterial saccharide conjugates optionally comprise N. meningitidis serogroup C capsular saccharide (MenC), serogroup C and Y capsular saccharides (MenCY), serogroup C and A capsular saccharides (MenAC), serogroup C and W capsular saccharides (MenCW), serogroup A and Y capsular

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WO 2007/000322 PCT/EP2006/006210 saccharide (MenAY), serogroup A and W capsular saccharides (MenAW), serogroup W and Y capsular saccharides (Men WY), serogroup A. C and W capsular saccharide (MenACW), serogroup A, C and Y capsular saccharides (MenACY); serogroup A. W135 and Y capsular saccharides (MenAWY), serogroup C, W135 and Y capsular saccharides (MenCWY); or serogroup A, C, W135 and Y capsular saccharides (MenACWY), serogroup B and C capsular saccharides (MenBC), serogroup B, C and Y capsular saccharides (MenBCY), serogroup B, C and A capsular saccharides (MenABC), serogroup B, C and W capsular saccharides (MenBCW), serogroup A. B and Y capsular saccharide (MenABY), serogroup A, B and W capsular saccharides (MenABW), serogroup B, W and Y capsular saccharides (MenBWY), serogroup A, B, C and W capsular saccharide (MenABCW), serogroup A, B. C and Y capsular saccharides (MenABCY); serogroup A, B, W135 and Y capsular saccharides (MenABWY), serogroup B, C, W135 and Y capsular saccharides (MenBCWY); or serogroup A, B, C, W135 and Y capsular saccharides (MenABCWY).

The immunogenic composition of the invention optionally contains the Hib saccharide conjugate in a saccharide dose between 0.1 and 9 pg; 1 and 5gg or 2 and 3gg or around or exactly 2.5pg and each of the at least two further saccharide conjugates at a dose of between 2 and 20gg, 3 and lOgg, or between 4 and 7pg or around or exactly 5gg.

“Around’ or “approximately” are defined as within 10% more or less of the given figure for the purposes of the invention.

The immunogenic composition of the invention contains a saccharide dose of the Hib saccharide conjugate which is for example less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the mean saccharide dose of the at least two further saccharide conjugates. The saccharide dose of the Hib saccharide is for example between 20% and 60%, 30% and 60%, 40% and 60% or around or exactly 50% of the mean saccharide dose of the at least two further saccharide conjugates.

The immunogenic composition of the invention contains a saccharide dose of the Hib saccharide conjugate which is for example less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the lowest saccharide dose of the at least two further saccharide conjugates. The saccharide dose of the Hib saccharide is for example between 20% and 60%, 30% and 60%, 40% and 60% or around or exactly 50% of the lowest saccharide dose of the at least two further saccharide conjugates.

In an embodiment of the invention, the dose of each of the two or more further saccharides is optionally the same, or approximately the same.

Examples of immunogenic compositions of the invention are compositions consisting of or comprising:

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Hib conjugate and MenA conjugate and MenC conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose of MenA is greater than the saccharide dose of MenC.

Hib conjugate and MenC conjugate and MenY conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1. 1:4:2, 1:4:1, 1:8;4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose of MenC is greater than the saccharide dose of MenY.

Hib conjugate and MenC conjugate and MenW conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8;4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenC is greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenW conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose of MenA is greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenY conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenA is greater than the saccharide dose of MenY.

Hib conjugate and MenW conjugate and MenY conjugate, optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:1:2, 1:4:2, 1:2:4, 1:4:1, 1:1:4, 1:3;6, 1:1:3, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenY is greater than the saccharide dose of MenW.

Hib and at least two further saccharides included in pharmaceutical compositions of the invention are conjugated to a carrier protein such as tetanus toxoid, tetanus toxoid fragment C, non-toxic mutants of tetaus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chern. 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, Gin or Ser and/or Ala 158 to Gly and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5917017 or US 6455673; or fragment disclosed in US 5843711], pneumococcal pneumolysin, 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) pneumolysin (Kuo et al (1995)

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Infect Immun 63; 2706-13), iron uptake proteins (WO 01/72337), toxin A or B of C. difficile QNO 00/61761) or Protein D (US6342224).

In an embodiment, the immunogenic composition of the invention uses the same carrier protein (independently selected) in the Hib conjugate and the at least two further bacterial saccharide conjugates, optionally in the Hib conjugate and each of the at least two further bacterial saccharide conjugates (e.g. all the other saccharide conjugates present in the immunogenic composition).

In an embodiment, the immunogenic composition optionally comprises a Hib saccharide conjugate and MenA polysaccharide conjugate, a Hib saccharide conjugate and MenC polysaccharide conjugate, a Hib saccharide conjugate and MenW polysaccharide conjugate, a Hib saccharide conjugate and MenY polysaccharide conjugate, a Hib saccharide conjugate and MenA and MenC polysaccharide conjugates, a Hib saccharide conjugate and MenA and MenW polysaccharide conjugates, a Hib saccharide conjugate and MenA and MenY polysaccharide conjugates, a Hib saccharide conjugate and MenC and MenW polysaccharide conjugates, a Hib saccharide conjugate and MenC and MenY polysaccharide conjugates, a Hib saccharide conjugate and MenW and MenY polysaccharide conjugates, a Hib saccharide conjugate and MenA, MenC and MenW polysaccharide conjugates, a Hib saccharide conjugate and MenA, MenC and MenY polysaccharide conjugates, a Hib saccharide conjugate and MenA, MenW and MenY polysaccharide conjugates, a Hib saccharide conjugate and MenC, MenW and MenY polysaccharide conjugates or a Hib saccharide conjugate and MenA, MenC, MenW and MenY polysaccharide conjugates.

In an embodiment, a single carrier protein may carry more than one saccharide antigen (WO 04/083251). For example, a single carrier protein might be conjugated to Hib and MenA, Hib and MenC, Hib and MenW, Hib and MenY, MenA and MenC, MenA and MenW, MenA and MenY, MenC and MenW, MenC and MenY or Men W and MenY.

In an embodiment, the immunogenic composition of the invention comprises a Hib saccharide conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197, fragment C of TT and protein D.

Where the carrier protein is TT or fragment thereof for Hib and the at least two further saccharides, the total dose of earner is between 2.5-25gg , 3-20pg, 4-15gg, 5-12.5gg, 1520gg, 16-19pg or 17-18gg.

In an embodiment, the immunogenic composition of the invention comprises at least two further bacterial saccharides conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197, fragment C of TT and protein D.

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The immunogenic composition of the invention optionally comprises a Hib saccharide conjugate having a ratio of Hib to carrier protein of between 1:5 and 5:1; 1:2 and 2:1; 1:1 and 1:4; 1:2 and 1:3.5; or around or exactly 1:2.5 or 1:3 (w/w).

The immunogenic composition of the invention optionally comprises at least one meningococcal saccharide (for example MenA and/or MenC and/or MenW and/or MenY) conjugate having a ratio of Men saccharide to carrier protein of between 1:5 and 5:1, between 1:2 and 5:1, between 1:0.5 and 1:2.5 or between 1:1.25 and 1:2.5(w/w).

The ratio of saccharide to carrier protein (w/w) in a conjugate may be determined using the sterilized conjugate. The amount of protein is determined using a Lowry assay ( for example Lowry et al (1951) J. Biol. Chern. 193, 265-275 or Peterson et al Analytical Biochemistry 100, 201-220 (1979)) and the amount of saccharide is determined using ICP-OES (inductively coupled plasma-optical emission spectroscopy) for MenA, DMAP assay for MenC and Resorcinol assay for MenW and MenY (Monsigny et al (1988) Anal. Biochem. 175, 525-530).

In an embodiment, the immunogenic composition of the invention the Hib saccharide is conjugated to the carrier protein via a linker, for instance a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, having for example a reactive amino group and a reative carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The linker has for example between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible linker is ADH. Other linkers include B-propionamido (WO 00/10599), nitrophenyl-ethylamine (Gever et al (1979) Med. Microbiol. Immunol. 165; 171288), haloalkyl halides (US4057685) glycosidic linkages (US4673574, US4808700) and 6aminocaproic acid (US4459286).

The saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique. For example the saccharide can be coupled via a thioether linkage. The conjugation method may rely on activation 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. Optionally, the cyanate ester is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using heteroligation chemistry involving the formation of the thioether linkage, or is conjugated to the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry. Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094.

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Other suitable techniques use carbiinides, hydrazides, active esters, norborane, pnitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98/42721. Conjugation may involve a carbonyl linker which may be formed by reaction of a free hydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction of with a protein to form a carbamate linkage. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group* reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate with an amino group on a protein.

The conjugates can also be prepared by direct reductive amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP) activated saccharide derivatised with adipic acid hydrazide (ADH) to the protein carrier by carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256), for example using EDAC.

In an embodiment, a hydroxyl group on a saccharide is linked to an amino or carboxylic group on a protein either directly or indirectly (through a linker). Where a linker is present, a hydroxyl group on a saccharide is optionally linked to an amino group on a linker, for example by using CDAP conjugation. A further amino group in the linker for example ADH) may be conjugated to a carboxylic acid group on a protein, for example by using carbodiimide chemistry, for example by using EDAC. In an embodiment, the Hib or at least two further saccharides is conjugated to the linker first before the linker is conjugated to the carrier protein.

In an embodiment, the Hib saccharide is conjugated to the carrier protein using CNBr, or CDAP, or a combination of CDAP and carbodiimide chemistry (such as EDAC), or a combination of CNBr and carbodiimide chemistry, (such as EDAC). Optionally Hib is conjugated using CNBr and carbodiimide chemistry (such as EDAC). For example, CNBr is used to join the saccharide and linker and then carbodiimide chemistry is used to join linker to the protein carrier.

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In an embodiment, at least one of the at least two further saccharides is directly conjugated to a carrier protein, optionally Men W and/or MenY and/or MenC saccharide(s) is directly conjugated to a carrier protein. For example MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC are directly linked to the carrier protein. Optionally at least one of the at least two further saccharides is directly conjugated by CDAP. For example MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC are directly linked to the carrier protein by CDAP (see WO 95/08348 and WO 96/29094).

In an embodiment, the ratio of Men W and/or Y saccharide to carrier protein is between 1:0.5 and 1:2 (w/w) or the ratio of MenC saccharide to carrier protein is between 1:0.5 and 1:2 or 1:1.25 and 1:1.5 or 1:0.5 and 1:1 (w/w), especially where these saccharides are directly linked to the protein, optionally using CDAP.

In an embodiment, at least one of the at least two further saccharides is conjugated to the carrier protein via a linker, for instance a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, having for example a reactive amino group and a reative carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The linker has for example between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible linker is ADH.

In an embodiment, MenA; MenC; or MenA and MenC is conjugated to a carrier protein via a linker.

In an embodiment, the further saccharide is conjugated to a carrier protein via a linker using CDAP and EDAC. For example, MenA; MenC; or MenA and MenC are conjugated to a protein via a linker (for example those with two amino groups at its ends such as ADH) using CDAP and EDAC as described above. For example, CDAP is used to conjugate the saccharide to a linker and EDAC is used to conjugate the linker to a protein. Optionally the conjugation via a linker results in a ratio of saccharide to carrier protein of of between 1:0.5 and 1:6; 1:1 and 1:5 or 1:2 and 1:4, for MenA; MenC; or MenA and MenC.

In an embodiment of the invention, the immunogenic composition comprises N. meningitidis capsular polysaccharides from at least one, two, three or four of serogroups A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four or each N. meningitidis polysaccharide is either a native polysaccharide or is sized by a factor up to x2, x3, x4, x5, x6, x7, x8, x9 , x10 or x20. For example, the average size of at least one, two, three or four or each N. meningitidis polysaccharide is above 50kDa, 60kDa, 75kDa, 100kDa, 110kDa, 120kDaor 130kDa.

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WO 2007/000322 PCT/EP2006/006210 “Native polysaccharide refers to a polysaccharide that has not been subjected to a process, the purpose of which is to reduce the size of the polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N. meningitidis capsular polysaccharides from at least one, two, three or four of serogroups A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four or each N. meningitidis polysaccharide is native polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N. meningitidis capsular polysaccharides from at least one, two, three or four of serogroups A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four or each N. meningitidis polysaccharide is sized by a factor up to x2, x3, x4, x5, x6, x7, x8, x9 or x10.

In an embodiment, the mean size of at least one, two, three, four or each N. meningitidis polysaccharide, where present, is between 50KDa and 1500kDa, 50kDa and 500kDa, 50 kDa and 300 KDa, 101 kDa and 1500kDa, 101 kDa and 500kDa, 101 kDa and 300kDa as determined by MALLS.

In an embodiment, the MenA saccharide, where present, has a molecular weight of 50500kDa, 50-100kDa, 100-500kDa, 55-90KDa, 60-70kDa or 70-80kDa or60-80kDa.

In an embodiment, the MenC saccharide, where present, has a molecular weight of 100200kDa, 50-100kDa, 100-150kDa, 101-130kDa, 150-210kDa or 180-210kDa.

In an embodiment the MenY saccharide, where present, has a molecular weight of 60190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa or 110-140kDa, 50-100kDa, 100-140kDa, 140-170kDa or 150-l60kDa.

In an embodiment the MenW saccharide, where present, has a molecular weight of 60190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa, 110-140kDa, 50-100kDa or 120-140kDa.

The molecular weights of the saccharide refers to the molecular weight of the polysaccharide measured prior to conjugation and is measured by MALLS.

In an embodiment any N. meningitidis saccharides present are native polysaccharides or native polysaccharides which have reduced in size during a normal extraction process.

In an embodiment, any N. meningitidis saccharides present are sized by mechanical cleavage, for instance by microfluidisation or sonication. Microfluidisation and sonication have the advantage of decreasing the size of the larger native polysaccharides sufficiently to provide a filterable conjugate.

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In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or 11.05 and after conjugation to a carrier protein, the polydispersity of the conjugate is 1.02,5, 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.

For MALLS analysis of meningococcal saccharides, two columns (TSKG6000 and 5000PWxl TOSOH Bioscience) 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 10mW argon laser at 488nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm).

In an embodiment, the MenA saccharide, where present is is at least partially Oacetylated such that at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one position. O-acetylation is for example present at least at the O-3 position.

In an embodiment, the MenC saccharide, where present is is at least partially Oacetylated such that at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of (a2 —*9)-linked NeuNAc repeat units are O-acetylated at at least one or two positions. Oacetylation is for example present at the O-7 and/or O-8 position.

In an embodiment, the MenW saccharide, where present is is at least partially Oacetylated such that at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one or two positions. O-acetylation is for example present at the O-7 and/or O-9 position.

In an embodiment, the MenY saccharide, where present is at least partially O-acetylated such that at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one or two positions. O-acetylation is present at the 7 and/or 9 position.

The percentage of O-acetylation refers to the percentage of the repeat units containing Oacetylation. This may be measured in the saccharide prior to conjugate and/or after conjugation.

A further aspect of the invention is a vaccine comprising the immunogenic composition of the invention and a pharmaceutically acceptable excipient.

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Optionally, the immunogenic composition or vaccine 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, polphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875.

For the HibMen combinations discussed above, it may be advantageous not to use any aluminium salt adjuvant or any adjuvant at all.

In an embodiment, the immunogenic composition comprises a Hib saccharide conjugated to tetanus toxoid via a linker and MenC saccharide conjugated to tetanus toxoid either directly or through a linker and MenY saccharide conjugated to tetanus toxoid.

In an embodiment, the immunogenic composition of the invention is 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.

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. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

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 one which will provide a single therapeutically effective dosage. For bioactive

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WO 2007/000322 PCT/EP2006/006210 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 1100 pg of protein antigen, for example 5-50pg or 5-25 pg. In an embodiment, doses of individual bacterial saccharides are 10-20pg, 10-5pg, 5-2.5pg or 2.5-1 pg. The preferred amount of the substance varies from substance to substance but is easily determinable by one of skill in the art.

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. 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 coadministered 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, US Patent 4,235,877.

A further aspect of the invention is a vaccine kit for concomitant or sequential administration comprising two multi-valent immunogenic compositions for conferring protection in a host against diease caused by Bordetelia pertussis, Clostridium tetani,

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Corynebacterium diphtheriae, Haemophilus influenzae and Neisseria meningitidis. For example, the kit optionally comprises a first container comprising one or more of:

tetanus toxoid (TT), diphtheria toxoid (DT), and whole cell or acellular pertussis components and a second container comprising either:

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates, wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of all the at least two further bacterial saccharide conjugates;

or

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates, wherein the Hib conjugate is present in a lower saccharide dose than each of the at least two further bacterial saccharide conjugates (e.g. at a lower saccharide dose that any saccharide present in the composition).

Examples of the Hib conjugate and the at least two further bacterial saccharide conjugates are as described above. '

A further aspect of the invention is a vaccine kit for concomitant or sequential administration comprising two multi-valent immunogenic compositions for conferring protection in a host against diease caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. For example, the kit optionally comprises a first container comprising:

one or more conjugates of a carrier protein and a capsular saccharide from Streptococcus pneumoniae [where the capsular saccharide(s) is/are optionally from a pneumococcal serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].

and a second container comprising either:

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates,

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WO 2007/000322 PCT/EP2006/006210 wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of all the at least two further bacterial saccharide conjugates;

or

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates, wherein the Hib conjugate is present in a lower saccharide dose than each of the at least two further bacterial saccharide conjugates (e.g. at a lower saccharide dose that any saccharide present in the composition).

Examples of the Hib conjugate and the at least two further bacterial saccharide conjugates are as described above.

Typically the Streptococcus pneumoniae vaccine in the vaccine kit of the present invention will comprise polysaccharide antigens (optionally conjugated), wherein the polysaccharides are derived from at least four serotypes of pneumococcus chosen from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. Optionally the four serotypes include 6B, 14, 19F and 23F. More optionally, at least 7 serotypes are included in the composition, for example those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Optionally more than 7 serotypes are included in the composition, for instance at least 10,11, 12,13 or 14 serotypes. For example the composition in one embodiment includes 11 capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (optionally conjugated). In an embodiment of the invention at least 13 polysaccharide antigens (optionally conjugated) are included, although further polysaccharide antigens, for example 23 valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention.

The pneumococcal saccharides are conjugated to any known carrier protein, for example CRM197, tetanus toxoid, diphtheria toxoid, protein D or any other carrier proteins as mentioned above.

Optionally, the vaccine kits of the invention comprise a third component. For example, the kit optionally comprises a first container comprising one or more of:

tetanus toxoid (TT), diphtheria toxoid (DT), and

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WO 2007/000322 PCT/EP2006/006210 whole cell or acellular pertussis components and a second container comprising :

one or more conjugates of a carrier protein and a capsular saccharide from Streptococcus pneumoniae [where the capsular saccharide is optionally from a pneumococcal serotype selected from the group consisting of 1, 2, 3, 4, 5. 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F], and a third container comprising:

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates, wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of all the at least two further bacterial saccharide conjugates;

or

Hib saccharide conjugate, and at least two further bacterial saccharide conjugates, wherein the Hib conjugate is present in a lower saccharide dose than each of the at least two further bacterial saccharide conjugates (e.g. at a lower saccharide dose that any saccharide present in the composition).

Immunogenic compositions comprising meningococcal conjugates, for example HibMenC, HibMenAC, HibMenAW, HibMenAY, HibMenCW, HibMenCY, HibMenWY, MenAC, MenAW, MenAY, MenCW, MenCY, MenWY or MenACWY, including kits of similar composition to those described above, optionally comprise antigens from measles and/or mumps and/or rubella and/or varicella. For example, the meningococcal immunogenic composition contains antigens from measles, mumps and rubella or measles, mumps, rubella and varicella. In an embodiment, these viral antigens are optionally present in the same container as the meningococcal and/or Hib saccharide conjugate(s). In an embodiment, these viral antigens are lyophilised.

A further aspect of the invention is a process for making the immunogenic composition of the invention, comprising the step of mixing a Hib saccharide conjugate with at least two further bacterial saccharide conjugates to form a composition in which the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of the at least two further bacterial saccharide conjugates.

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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. US Patent 4,235,877.

A further aspect of the invention is a method of immunising a human host against disease caused by Haemophilus influenzae and optionally N. meningitidis infection comprising administering to the host an immunoprotective dose of the immunogenic composition or vaccine or kit of the invention.

A further aspect of the invention is an immunogenic composition of the invention for use in the treatment or prevention of disease caused by Haemophilus influenzae and optionally N. meningitidis.

A further aspect of the invention is use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of diseases caused by Haemophilus influenzae and optionally N. meningitidis.

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.

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.

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Examples

Example 1 - preparation of polysaccharide conjugates

The covalent binding of Haemophilus influenzae (Hib) PRP polysaccharide to TT was carried out by a coupling chemistry developed by Chu et al (Infection and Immunity 1983, 40 (1); 245-256). Hib PRP polysaccharide was activated by adding CNBr and incubating at pH10.5 for 6 minutes. The pH was lowered to pH8.75 and adipic acid dihydrzide (ADH) was added and incubation continued for a further 90 minutes. The activated PRP was coupled to purified tetanus toxoid via carbodiimide condensation using 1-ethyl-3-(3dimethyl-aminopropyl)carbodiimide (EDAC). EDAC was added to the activated PRP to reach a final ratio of 0.6mg EDAC/mg activated PRP. The pH was adjusted to 5.0 and purified tetanus toxoid was added to reach 2mg TT/mg activated PRP. The resulting solution was left for three days with mild stirring. After filtration through a 0.45gm membrane, the conjugate was purifed on a Sephacryl S500HR (Pharmacia, Sweden) column equilibrated in 0.2M NaCI.

MenC —TT conjugates were produced using native polysaccharides ( of over 150kDa as measured by MALLS). MenA-TT conjugates were produced using either native polysaccharide or slightly microfluidised polysaccharide of over 60kDa as measured by the MALLS method of example 2. MenW and MenY-TT conjugates were produced using sized polysaccharides of around 100-200kDa as measured by MALLS (see example 2). Sizing was by microfluidisation using a homogenizer Emulsiflex C-50 apparatus. The polysaccharides were then filtered through a 0.2pm filter.

Activation and coupling were performed as described in WO96/29094 and WO 00/56360. Briefly, the polysaccharide at a concentration of 10-20mg/ml in 2M NaCI pH 5.5-6.0 was mixed with CDAPsolution (100mg/ml freshly prepared in acetonitrile/WFI, 50/50) to a final CDAP/polysaccharide ratio of 0.75/1 or 1.5/1. After 1.5 minutes, the pH was raised with sodium hydroxide to pH 10.0. After three minutes tetanus toxoid was added to reach a protein/polysaccharide ratio of 1.5/1 for MenW, 1.2/1 for MenY, 1.5/1 for MenA or 1.5/1 for MenC. The reaction continued for one to two hours.

After the coupling step, glycine was added to a final ratio of glycine/PS (w/w) of 7.5/1 and the pH was adjusted to pH9.0. The mixture was left for 30 minutes. The conjugate was clarified using a 10pm Kleenpak filter and was then loaded onto a Sephacryl S400HR column using an elution buffer of 150mM NaCI, 10mM or 5mM Tris pH7.5. Clinical lots were filtered on an Opticap 4 sterilizing membrane. The resultant conjugates had an average polysaccharide:protein ratio of 1:1-1:5 (w/w).

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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 10mg/ml solution of MenA was treated with a freshly prepared 100mg/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.

Prior to the conjugation reaction, the purified TT solution and the PSAah solution were diluted to reach a concentration of 10 mg/ml for PSAah and 10mg/ml for TT.

EDAC was added to the PSah solution 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 conjugate was clarified using a 10gm filter and was purified using a Sephacryl S400HR column.

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 (M_w) 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-

b) Number-average molecular weight: -Mn-

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ΣΝ,. M, _m, Σ^Νι ^mo

c) Root mean square radius: -Rw- and R%/ is the square radius defined by:

R²w or (Ôw = -=^--(-mr is the mass of a scattering centre i and -n- 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. 25μ1 of the polysaccharide were loaded onto the column and was eluted with 0.75ml of filtered water. The polyaccharides are detected using a light scattering detector ( Wyatt Dawn DSP equipped with a 10mW argon laser at 488nm) and an inferometric refractometer ( Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm).

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 Phase II clinical trial on HibMenAC -TT conjugate vaccine mixed with DTPwHepB

Study design: Open, randomized (1:1:1:1:1), single centre study with five groups. The five groups received the following vaccination regimen respectively, at 6, 10 and 14 weeks of age.

. Tritanrix™-HepB/Hib-MenAC 2.5/2.5/2.5: henceforth referred to as 2.5/2.5/2.5 . Tritanrix™-HepB/Hib-MenAC 2.5/5/5: henceforth referred to as 2.5/5/5 . Tritanrix™-HepB/Hib-MenAC 5/5/5: henceforth referred to as 5/5/5 . Tritanrix™-HepB + Hiberix™: henceforth referred to as Hiberix . Tritanrix™-HepB/Hiberix™ + Meningitec™: henceforth referred to as Meningitec

Blood samples were taken at the time of the first vaccine dose (Pre) and one month after the third vaccine dose (Post-dose 3).

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Tritanrix is a DTPw vaccine marketted by GlaxoSmithKline Biologicals S.A.

105 subjects were used in each of the five groups giving a total of 525 subjects in the study.