HK1116082B - Immunogenic composition - Google Patents

Description

The present invention relates to immunogenic compositions comprising N. meningitidis capsular polysaccharides conjugated to a carrier protein. It additionally relates to vaccines and vaccine kits comprising N. meningitidis polysaccharide conjugates, processes for making the immunogenic compositions and vaccines and the use of the vaccines and immunogenic compositions of the invention in therapy. It also relates to the immunogenic compositions provided for use in methods of immunising against Neisserial infection.

Neisseria meningitidis is a Gram-negative human pathogen which causes bacterial meningitis. Based on the organism's capsular polysaccharide, twelve serogroups of N. meningitidis have been identified (A, B, C, H, I, K, L, 29E, W135, X, Y and Z). Serogroup A (MenA) is the most common cause of epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the majority of cases in developing countries, with the remaining cases being caused by W135 and Y).

Immunogenic compositions comprising N. meningitidis saccharides conjugated to carrier proteins are known in the art. For instance WO 02/58737 discloses a vaccine comprising purified capsular polysacchrides from N. meningitidis serogroups A, C, W135 and Y conjugated to a carrier protein. However, this application teaches that the extracted N. meningitidis capsular polysaccharides should be depolymerised by heating in a hydrogen peroxide solution before conjugation.

WO 03/07985 discloses conjugate vaccines comprising N. meningitidis saccharide selected from serogroups A, C, W135 and Y. The meningococcal capsular polysaccharides are extracted and then hydrolysed so that a selection of oligosaccharides derived from the capsular polysaccharides are used for conjugation to a carrier protein.

WO 04/103400 also discloses multivalent meningococcal derived polysaccharide-protein conjugate containing capsular polysaccharides derived from N. meningitidis serogroups A, C, W135 and Y. This application teaches that, instead of using the large native capsular polysaccharide, the use of meningococcal polysaccharides of a smaller size is preferred. It suggests that capsular polysaccharides are partially depolymerised using mild oxidative conditions to give an average size of less than 100,000 daltons, preferably 12,000 to 25,000 daltons.

There remains a need to develop improved conjugate vaccines against neisserial meningitis. The present invention concerns the provision of a meningococcal polysaccharide conjugate vaccine in which the size of the polysaccharides is larger than that taught in the literature. The focus of the art has been to use oligosaccharides for ease of conjugate production. The inventors has found that by using native or slightly sized polysaccharide conjugates, one or more of the following advantages may be realised: 1) a conjugate having high immungenicity which is filterable; 2) immune memory may be enhanced (as in example three); 3) the alteration of the ratio of polysaccharide to protein in the conjugate such that the ratio of polysaccharide to protein (w/w) in the conjugate may be increased (this can result in a reduction of the carrier suppression effect); 4) immunogenic conjugates prone to hydrolysis (such as MenA conjugates) may be stabilised by the use of larger polysaccharides for conjugation. The use of larger polysaccharides can result in more cross-linking with the conjugate carrier and therefore less cleavage of free saccharide from the conjugate. The conjugate vaccines described in the prior art tend to depolymerise the polysaccharides prior to conjugation in order to improve conjugation. The present invention is directed to a different strategy and surprisingly shows that meningococcal conjugate vaccines retaining a larger size of polysaccharide provide a good immune response against meningococcal disease.

Accordingly, in one aspect of the present invention there is provided an immunogenic composition that either does not use any aluminium salt adjuvant or does not use any adjuvant at all, the composition comprising N. meningitidis capsular polysaccharides from at least one of serogroups A, C, W135 and Y conjugated to a carrier protein to produce a N. meningitidis capsular polysaccharide conjugate, wherein the average size of each N. meningitidis polysaccharide is above 50kDa, wherein each N. meningitidis polysaccharide is conjugated to tetanus toxoid carrier and is either a native polysaccharide or is sized by microfluidization, wherein said composition comprises a MenC capsular polysaccharide conjugated to tetanus toxoid and said MenC capsular polysaccharide has an average size of 150-210 kDa.

According to a further aspect or the invention there is provided a vaccine comprising the immunogenic composition of the invention and a pharmaceutically acceptable carrier.

According to a further aspect or the invention there is provided a vaccine kit for concomitant or sequential administration comprising two multi-valent immunogenic compositions for conferring protection in a host against disease caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Haemophilus influenzae and Neisseria meningitidis, said kit comprising a first container comprising :

• tetanus toxoid (TT),
• diphtheria toxoid (DT), and
• wholecell or acellular pertussis components
• and a second container comprising: the immunogenic composition of the invention.

According to a further aspect of the invention there is provided a process for making the vaccine of the invention comprising the step of mixing the immunogenic composition of the invention with a pharmaceutically acceptable carrier.

According to a further aspect or the invention there is provided an immunogenic composition of the invention for use in the treatment or prevention of disease caused by Neisseria meningitidis.

According to a further aspect or the invention there is provided a use of the immunogenic composition or vaccine of the invention in the manufacture of a medicament for the treatment or prevention of diseases caused by Neisseria meningitidis.

Description of figures

Figure 1 - A - Bar chart showing GMC responses in an anti-MenY ELISA. ENYTT012 is a MenY-TT conjugate prepared from native MenY polysaccharide. ENYTT014 is a MenY-TT conjugate prepared from microfluidised MenY polysaccharide which had undergone 40 cycles of microfluidisation. ENYTT015bis is a MenY-TT conjugate prepared from microfluidised MenY polysaccharide which had undergone 20 cycles of microfluidisation.

- B - Bar chart showing GMT responses in an anti-MenY SBA assay. ENYTT012 is a MenY-TT conjugate prepared from native MenY polysaccharide. ENYTT014 is a MenY-TT conjugate prepared from microfluidised MenY polysaccharide which had undergone 40 cycles of microfluidisation. ENYTT015bis is a MenY-TT conjugate prepared from microfluidised MenY polysaccharide which had undergone 20 cycles of microfluidisation.

Detailed description

An immunogenic composition of the invention is one that either does not use any aluminium salt adjuvant or does not use any adjuvant at all, the composition comprising N. meningitidis capsular polysaccharides from at least one of serogroups A, C, W135 and Y conjugated to a carrier protein to produce a N. meningitidis capsular polysaccharide conjugate, wherein the average size of each N. meningitidis polysaccharide is above 50kDa, wherein each N. meningitidis polysaccharide is conjugated to tetanus toxoid carrier and is either a native polysaccharide or is sized by microfluidization, wherein said composition comprises a MenC capsular polysaccharide conjugated to tetanus toxoid and said MenC capsular polysaccharide has an average size of 150-210 kDa. The immunogenic composition of the invention may comprise 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 the average size (weight-average molecular weight; Mw) of at least one, two, three or four or each N. meningitidis polysaccharide is above 50kDa.

Also disclosed is an immunogenic composition that 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 x1.5, x2, x3, x4, x5, x6, x7, x8, x9 or x10 relative to the weight average molecular weight of the native polysaccharide. An immunogenic composition of the invention comprises a MenC capsular polysaccharide conjugated to tetanus toxoid and said MenC capsular polysaccahraide has an average size of 150-210kDa.

For the purposes of the invention, "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. A polysaccharide can become slightly reduced in size during normal purification procedures. Such a polysaccharide 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 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.

Also disclosed is an immunogenic composition that 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.

Also disclosed is an immunogenic composition that 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 x1.5, x2, x3, x4, x5, x6, x7, x8, x9 or x10.

Also disclosed are immunogenic compositions that optionally comprise conjugates of : N. meningitidis serogroup C capsular polysaccharide (MenC), serogroup A capsular polysaccharide (MenA), serogroup W135 capsular polysaccharide (MenW), serogroup Y capsular polysaccharide (MenY), serogroup C and Y capsular polysaccharides (MenCY), serogroup C and A capsular polysaccharides (MenAC), serogroup C and W capsular polysaccharides (MenCW), serogroup A and Y capsular polysaccharide (MenAY), serogroup A and W capsular polysaccharides (MenAW), serogroup W and Y capsular polysaccharides (Men WY), serogroup A, C and W capsular polysaccharide (MenACW), serogroup A, C and Y capsular polysaccharides (MenACY); serogroup A, W135 and Y capsular polysaccharides (MenAWY), serogroup C, W135 and Y capsular polysaccharides (MenCWY); or serogroup A, C, W135 and Y capsular polysaccharides (MenACWY). This is the definition of "one, two, three or four", or "at least one of" of serogroups A, C, W and Y, or of each N. meningitidis polysaccharide where mentioned herein

In an embodiment, the average size (or molecular weight) of at least one, two, three, four or each N. meningitidis polysaccharide is 50KDa - 1500kDa, 50kDa - 500kDa, 50 kDa - 300 KDa, 101kDa - 1500kDa, 101kDa - 500kDa, or 101kDa - 300kDa as determined by MALLS.

In an embodiment, the MenA polysaccharide, where present, has a molecular weight of 50-500kDa, 50-100kDa, 100-500kDa, 55-90KDa, 60-70kDa or 70-80kDa or 60-80kDa as determined by MALLS.

An immunogenic composition of the invention comprises a MenC capsular polysaccharide conjugated to tetanus toxoid and said MenC capsular polysaccahraide has an average size of 150-210kDa. In an embodiment, the MenC polysaccharide has a molecular weight of 180-210kDa as determined by MALLS.

In an embodiment the MenY polysaccharide, where present, has a molecular weight of 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa or 110-140kDa, 50-100kDa, 100-140kDa, 140-170kDa or 150-160kDa as determined by MALLS.

In an embodiment the MenW polysaccharide, where present, has a molecular weight of 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa, 110-140kDa, 50-100kDa or 120-140kDa as determined by MALLS.

The molecular weight or average molecular weight of a polysaccharide herein refers to the weight-average molecular weight (Mw) of the polysaccharide 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 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 N. meningitidis polysaccharides are native polysaccharides or native polysaccharides which have reduced in size during a normal extraction process.

In an embodiment, the N. meningitidis polysaccharides 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. Sizing is by a factor of no more than x20, x10, x8, x6, x5, x4, x3 , x2 or x1.5.

Also described is an immunogenic composition that comprises N. meningitidis conjugates that are made from a mixture of native polysaccharides and polysaccharides that are sized by a factor of no more than x20. For example, polysaccharides from MenC and/or MenA are native. For example, polysaccharides from MenY and/or MenW are sized by a factor of no more than x20, x10, x8, x6, x5, x4, x3 , x2 or x1.5. For example, an immunogenic composition contains a conjugate made from MenY and/or MenW and/or MenC and/or MenA which is sized by a factor of no more then x20, x10, x8, x6, x5, x4, x3 , x2 or x1.5 and/or is microfluidised. For example, an immunogenic composition contains a conjugate made from native MenA and/or MenC and/or MenW and/or MenY. For example, an immunogenic composition comprises a conjugate made from native MenC. For example, an immunogenic composition comprises a conjugate made from native MenC and MenA which is sized by a factor of no more than x20, x10, x8, x6, x5, x4, x3 , x2 or x1.5 and/or is microfluidised. For example, an immunogenic composition comprises a conjugate made from native MenC and MenY which is sized by a factor of no more than x20, x10, x8, x6, x5, x4, x3 , x2 or x1.5 and/or is microfluidised.

In an embodiment, the polydispersity of the polysaccharide 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.

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.

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 immunogenic composition of the invention further comprises a H. influenzae b (Hib) capsular saccharide conjugated to a carrier protein.

The N. meningitidis polysaccharide(s) (and optionally Hib capsular saccharide) included in pharmaceutical compositions of the invention are each conjugated to tetanus toxoid carrier. Optionally the Hib may be conjugated to tetanus toxoid fragment C, non-toxic mutants of tetanus toxin, diphtheria toxoid, 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 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) Infect Immun 63; 2706-13), iron uptake proteins ( WO 01/72337 ), toxin A or B of C. difficile ( WO 00/61761 ) or Protein D ( EP594610 and WO 00/56360 ). In an immunogenic composition of the invention the MenC capsular polysaccharide is conjugated to tetanus toxoid.

The immunogenic composition of the invention uses Tetanus Toxoid for each N. meningitidis polysaccharide (independently). In an embodiment where Hib is present, Hib may be conjugated to the same carrier protein.

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 MenA and MenC; MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; MenA, MenC, MenW and MenY; Hib and MenA; Hib and MenC; Hib and MenW; or Hib 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.

Also disclosed is an immunogenic composition that comprises at least one meningococcal saccharide (for example MenA; MenC; MenW; MenY; MenA amd MenC; MenA and MenW; MenA and MenY; MenC and Men W; Men C and MenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MwnY; MenC, MenW and MenY or MenA, MenC, MenW and 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 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). By 'around' it is meant within 10% of the stated ratio.

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. Chem. 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 N. meningitidis polysaccharide(s) and/or 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 reactive 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; 171-288), haloalkyl halides ( US4057685 ), glycosidic linkages ( US4673574 , US4808700 ), hexane diamine and 6-aminocaproic acid ( US4459286 ).

The polysaccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique. 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. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a holoacetylated carrier protein (for example using iodoacetimide or N-succinimidyl bromoacetatebromoacetate). Optionally, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using 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 .

Other suitable techniques use carbiinides, hydrazides, active esters, norborane, p-nitrobenzoic 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 (optionally an activated hydroxyl group for example a hydroxyl group activated by a cyanate ester) 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 N. meningitidis capsular polysaccharide(s) is conjugated to the linker first before the linker is conjugated to the carrier protein.

In an embodiment, the Hib saccharide, where present, 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, optionally 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.

In an embodiment, at least one of the N. meningitidis capsular polysaccharides 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 the at least one of the N. meningitidis capsular polysaccharides 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, all N. meningitidis capsular polysaccharides are conjugated to tetanus toxoid.

Optionally the ratio of Men W and/or Y saccharide to carrier protein is between 1:0.5 and 1:2 (w/w) and/or the ratio of MenC saccharide to carrier protein is between 1:0.5 and 1:4 or 1:1.25-1:1.5 or 1:0.5 and 1:1.5 (w/w), especially where these saccharides are directly linked to the protein, optionally using CDAP.

In an embodiment, at least one of the N. meningitidis capsular polysaccharide(s) 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 amine group and a reative carboxylic acid group, 2 reactive amine groups or 2 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 (for example tetanus toxoid) via a linker.

In an embodiment, at least one N. meningitidis polysaccharide 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 hydrozino 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 polysaccharide 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, the MenA capsular polysaccharide, where present is is at least partially O-acetylated 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 of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

In an embodiment, the MenC capsular polysaccharide is at least partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of (α2 →9)-linked NeuNAc 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-8 position of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

In an embodiment, the MenW capsular polysaccharide, where present is is at least partially O-acetylated 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 of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

In an embodiment, the MenY capsular polysaccharide, where present is at least partially O-acetylated such that at least 20%, 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 present at the 7 and/or 9 position of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

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

In a further embodiment, the immunogenic composition of the invention comprises a Hib saccharide conjugate and at least two N. meningitidis polysaccharide conjugates wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of the at least two N. meningitidis polysaccharide conjugates. Alternatively, the Hib conjugate is present in a lower saccharide dose than the saccharide dose of each of the at least two N. meningitidis polysaccharide 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 N. meningitidis polysaccharide 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 are characterised by typically being hydrolysed polysaccharides with a low number of repeat units (typically 5-30 repeat units).

The mean dose is determined by adding the doses of all the further polysaccharides and dividing by the number of further polysaccharides. Further polysaccharides are all the polysaccharides within the immunogenic composition apart from Hib and can include N. meningitidis capsular polysaccharides. The "dose" is in the amount of immunogenic composition or vaccine that is administered to a human.

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

'At least two further bacterial saccharide conjugates' is to be taken to mean at least two further bacterial saccharide conjugates in addition to a Hib conjugate. The at least two further bacterial conjugates may include N. meningitidis capsular polysaccharide conjugates.

The immunogenic compositions of the invention may comprise further saccharide conjugates 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, 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 (optionally 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 Ic, Type II, Type III or Type V 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 and optionally multiple types of M protein.

In an embodiments, the immunogenic composition of the invention contains each N. meningitidis capsular polysaccharide at a dose of between 0.1-20µg; 1-10µg; 2-10µg, 2.5-5µg, around or exactly 5µg; or around or exactly 2.5µg.

In an embodiment, the immunogenic composition of the invention for example contains the Hib saccharide conjugate at a saccharide dose between 0.1 and 9µg; 1 and 5µg or 2 and 3µg or around or exactly 2.5µg and each of the N. meningitidis polysaccharide conjugates at a saccharide dose of between 2 and 20µg, 3 and 10µg, or between 4 and 7µg or around or exactly 5µg.

"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 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 at least two, three, four or each of the N. meningitidis polysaccharide 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 at least two, three, four or each of the N. meningitidis polysaccharide conjugates.

In an embodiment, 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, three, four or each of the N. meningitidis polysaccharide 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, three, four or each of the N. meningitidis polysaccharide conjugates.

In an embodiment of the invention, the saccharide dose of each of the at least two, three, four or each of the N. meningitidis polysaccharide conjugates is optionally the same, or approximately the same.

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

• 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.
• MenA, MenC, MenW and MenY at saccharide dose ratios of 1:1:1:1 or 2:1:1:1 or 1:2:1:1 or 2:2:1:1 or 1:3:1:1 or 1:4:1:1 (w/w).

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

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.

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 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.

For the N. meningitidis or HibMen combinations discussed above, it may be advantageous not to use any adjuvant at all. The immunogenic composition of the invention does not comprise an aluminium salt adjuvant.

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 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 1-100µg of protein antigen, optionally 5-50µg or 5-25µg. Examples of doses of bacterial saccharides are 10-20µg, 5-10µg, 2.5-5µg or 1-2.5µg. 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 for use in methods 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 (12-25, 14-21 or 15-19 years) or an adult (any age over 12, 15, 18 or 21). 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, 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 disease caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae and Neisseria meningitidis and optionally Haemophilus influenzae. 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: an immunogenic composition of the invention, optionally lyophilised.orHib saccharide conjugate, andan immunogenic composition of the invention, optionally lyophilised.

Formulation examples of the Hib conjugate and the N. meningitidis polysaccharide 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 and Neisseria meningitidis and optionally Haemophilus influenzae. 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 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 second container comprising either: an immunogenic composition of the invention, optionally lyophilised.orHib saccharide conjugate, andan immunogenic composition of the invention, optionally lyophilised.

Examples of the Hib conjugate and the N. meningitidis polysaccharide conjugates are as described above.

Typically the Streptococcus pneumoniae vaccine in the vaccine kit of the present invention will comprise saccharide 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. 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 (optoinally 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, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention.

Also disclosed is a vaccine kit comprising a third component. 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 : 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: an immunogenic composition of the invention, optionally lyophillised.orHib saccharide conjugate, andan immunogenic composition of the invention, optionally lyophilised.

Immunogenic compositions of the invention may optionally further 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 vaccine of the invention comprising the step of mixing the immunogenic composition of the invention with a pharmaceutically acceptable carrier,.

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 an immunogenic composition of the invention for use in a method of immunising a human host against disease caused by N. meningitidis and optionally Haemophilus influenzae 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 N. meningitidis and optionally Haemophilus influenzae infection.

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

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 dihyrazide (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-(3-dimethylaminopropyl)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 0.45µm membrane, the conjugate was purifed on a sephacryl S500HR (Pharmacia, Sweden) column equilibrated in 0.2M NaCl.

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 0.2µm 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 NaCl 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 pH10.0. 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 10µm Kleenpak filter and was then loaded onto a Sephacryl S400HR column using an elution buffer of 150mM NaCl, 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).

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 PSA_AH solution were diluted to reach a concentration of 10 mg/ml for PSA_AH and 10mg/ml for TT. EDAC was added to the PS_AH solution in order to reach a final ratio of 0.9 mg EDAC/mg PSA_AH. 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 PSA_AH. 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 10µm 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.

1. a) Weight-average molecular weight: -Mw-
2. b) Number-average molecular weight: -Mn-
3. c) Root mean square radius: -Rw- and R²w is the square radius defined by: (-mi- is the mass of a scattering centre i and -r_i- is the distance between the scattering centre i and the center of gravity of the macromolecule).
4. 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. 25µl 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 - clinical trial comparing immunisation with Meningitec or a larger sized MenC-TT conjugate

A phase II , open, controlled study was carried out to compare GSK Biologicals meningococcal serogroup C conjugate vaccine (MenC) with GSK Biological's Haemophilus influenzae b-meningococcal serogroup C conjugate vaccine (Hib-MenC) or Meningitec®. Each dose of Meningitec® contains 10µg of meningococcal serogroup C oligosaccharide conjugated to 15µg of CRM197 and is produced by Wyeth. The GSK MenC conjugates contained native polysaccharides of about 200kDa conjugated to tetanus toxoid (TT).

The study consisted of five groups, each planned to contain 100 subjects, allocated to two parallel arms as follws: In this present study, all subjects in both arms received one-fifth (1/5) of a dose of Mencevax™ ACWY and a concomitant dose of Infanrix™ hexa at 12-15 months of age (Study Month 0). Two blood samples were collected from all subjects (Study Month 0 and Study Month 1). Arm 1 consisted of four groups from a primary vaccination study who were primed at their age of 3, 4 and 5 months with the following vaccines:

• Group K: MenC (10 µg), non-adsorbed onto aluminium salts (non-ads), tetanus toxoid (TT) conjugate and Infanrix™ hexa (MenC10-TT + Infanrix™ hexa)
• Group L: Hib (10 µg)-MenC (10 µg), non-ads TT conjugate and Infanrix™ penta (Hib10-MenC10-TT + Infanrix™ penta)
• Group M: Hib (5 µg)-MenC (5 µg), non-ads, TT conjugate and Infanrix™ penta (Hib5-MenC5-TT + Infanrix™ penta)
• Group N: Meningitec™ and Infanrix™ hexa (Meningitec™ + Infanrix™ hexa)

The two Hib-MenC-TT vaccine groups (Groups L and M) were kept blinded in the booster study as to the exact formulation of the candidate vaccine. Arm 2-(Group O) consisted of age-matched subjects not previously vaccinated with a meningococcal serogroup C vaccine (naïve) but who had received routine pediatric vaccines according to the German Permanent Commission on Immunization.

Criteria for evaluation:

Immunogenicity: Determination of bactericidal antibody titers against meningococcal C (SBA-MenC) by a bactericidal test (cut-off: a dilution of 1:8) and ELISA measurement of antibodies against meningococcal serogroup C (assay cut-off: 0.3 µg/ml), the Hib polysaccharide PRP (assay cut-off: 0.15 µg/ml) and tetanus toxoid (assay cut-off: 0.1 IU/ml) in blood samples obtained prior to vaccination and approximately one month after vaccination in all subjects.

Statistical methods:

Demographics: Determination of mean age in months (with median, range and standard deviation [SD]), and racial and gender composition of the ATP and Total vaccinated cohorts.

Immunogenicity:

Two analyses of immunogenicity were performed based on the ATP cohort for immunogenicity (for analyses of immune memory and booster response) or the ATP cohort for safety (for analysis of persistence). These included: Evaluation of immune memory for MenC and booster response for Hib and Tetanus (before and one month after administration of 1/5 dose of the plain polysaccharide vaccine):

• Determination of geometric mean titers and concentrations (GMTs and GMCs) with 95% confidence intervals (95% Cl)
• Determination of the percentage of subjects with antibody titer/concentration above the proposed cutoffs with exact 95% Cl (seropositivity/seroprotection rates)
• Investigation of antibody titers/concentration after vaccination using reverse cumulative curves
• Computation of standardized asymptotic 95% Cl for the difference in seropositivity/seroprotection rate
• between the primed group (Groups K, L, M and N) and the unprimed group (Group O)
• Determination of the geometric mean of individual ratio of SBA-MenC titer over anti-PSC concentration, with 95% CI
• Determination of the 95% CI for the post-vaccination GMT/C ratio between the groups K, L, M and the control group N for anti-PRP and anti-tetanus and between each primed group (Groups K, L, M and N) and the unprimed group (Group O) for SBA-MenC and anti-PSC using an ANOVA model

Results

Antibody	Group	N	GMT/C	95% CL LL	95% CL UL
SBA-MenC	K -MenC-TT	71	3508.9	2580.1	4772.2
L - HibMenC	79	2530.1	1831.7	3494.7
M-HibMenC	81	5385.4	4425.0	6554.2
N -Meningitec	85	1552.6	1044.4	2307.9
O - Control	91	9.3	6.3	13.6
Anti-PSC	K -MenC-TT	70	28.10	22.59	34.95
L - HibMenC	71	30.01	24.09	37.38
M-HibMenC	76	34.58	29.10	41.09
N -Meningitec	78	16.59	12.98	21.21
O - Control	94	3.05	2.36	3.93

Group K: subjects primed with MenC10-TT + Infanrix. hexa; Group L: subjects primed with Hib10-MenC10-TT + Infanrix. penta; Group M: subjects primed with Hib5-MenC5-TT + Infanrix. penta; Group N: subjects primed with Meningitec. + Infanrix. hexa; Group O: control subjects (i.e. subjects not primed with MenC conjugate vaccine) N: number of subjects with available results Higher titres of antibodies against MenC and higher SBA titres were achieved by priming with the larger sized MenC polysaccharide conjugate vaccines (groups K, L and M) compared with the Meningitec oligosaccharide conjugate vaccine.

Group	Timing	N	GMR	LL	UL
K	Pre	70	49.470	34.939	70.044
Post	66	126.138	101.419	156.882
L	Pre	76	36.528	25.849	51.621
Post	70	90.200	70.153	115.975
M	Pre	77	51.298	36.478	72.139
Post	74	164.950	139.304	195.318
N	Pre	84	22.571	16.521	30.837
Post	76	90.168	67.757	119.991
O	Pre	3	91.634	0.651	12889.8
Post	87	2.708	1.767	4.149

In all four primed groups (Groups K, L, M and N), the GMR increased significantly from pre to post booster vaccination indicating the presence of antibody maturation and functionality. GMR in the Group M (primed with Hib5-MenC5-TT) was higher than in the Group N (primed with Meningitec™).

Endpoints	Group	N	%	Group	N	%	Difference	Value%
SBAMenC	K	79	88.6	N	91	80.2	N-K	-8.4
≥ 1:8	L	84	93.3	N	91	80.2	N-L	-3.1
M	85	87.1	N	91	80.2	N-M	-6.8
SBAMenC	K	79	65.8	N	91	51.6	N-K	-14.2
≥ 1:128	L	84	56.0	N	91	51.6	N-L	-4.3
M	85	64.7	N	91	51.6	N-M	-13.1
Anti-PSC	K	79	100.0	N	91	100.0	N-K	0.0
≥0.3µg/ml	L	84	100.0	N	91	100.0	N-L	0.0
M	88	98.9	N	91	100.0	N-M	1.1
Anti-PSC	K	79	72.2	N	91	81.3	N-K	9.2
≥2µg/ml	L	84	64.3	N	91	81.3	N-L	17.0
M	88	64.3	N	91	81.3	N-M	8.6
Anti-PRP	K	81	88.9	N	91	85.7	N-K	-3.2
≥0.15µg/ml	L	86	96.5	N	91	85.7	N-L	-10.8
M	90	98.9	N	91	85.7	N-M	-13.2
Anti-PRP	K	81	33.3	N	91	28.6	N-K	-4.8
≥1µg/ml	L	86	55.8	N	91	28.6	N-L	-27.2
M	90	74.4	N	91	28.6	N-M	-45.9
Anti-tetanus	K	81	100.0	N	91	96.7	N-K	-3.3
≥0.1 IU/ml	L	86	100.0	N	91	96.7	N-L	-3.3
M	90	100.0	N	91	96.7	N-M	-3.3

Group K: subjects primed with MenC10-TT + Infanrix. hexa; Group L: subjects primed with Hib10-MenC10-TT + Infanrix. penta; Group M: subjects primed with Hib5-MenC5-TT + Infanrix. penta; Group N: subjects primed with Meningitec. + Infanrix. hexa; N: number of subjects with available results Higher SBA titres against MenC were achieved by priming with the larger size of MenC (groups K, L and M) compared to priming with the MenC-oligosaccharide conjugate Meningitec.

Immune memory (ATP cohort for immunogenicity)

Administration of 1/5 dose of the plain polysaccharide ACWY vaccine elicited very high SBA-MenC titer in all four primed groups with 98.7-100% and 97.5-100% of subjects primed with a candidate vaccine regimen exhibiting titers ≥1:8 and ≥1:128, respectively. In the group primed with the Meningitec™ regimen, there was a trend for a lower percentage of subjects with titers ≥1:128 (91.8%). In comparison, 17.6% of unprimed subjects had SBA MenC titers ≥ 1:8 and ≥1:128.

Example 4 Phase II clinical trial on HibMenAC -TT conjugate vaccine mixed with DTPw-HepB

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 2.5µg/2.5µg/2.5µg: henceforth referred to as 2.5/2.5/2.5
• Tritanrix.-HepB/Hib-MenAC 2.5 µg /5 µg /5 µg: henceforth referred to as 2.5/5/5
• Tritanrix.-HepB/Hib-MenAC 5 µg /5 µg /5 µg: 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).

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

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

Components per dose (0.5ml)	2.5/2.5/2.5*	2.5/5/5	5/5/5
Hib capsular polysaccharide PRP conjugated to tetanus toxoid (TT)	2.5µg	2.5µg	5µg
2.5µg	5µg	5µg
2.5µg	5µg	5µg

* The 2.5/2.5/2.5 vaccine was a dose dilution of GSK Biologicals' Hib-MenAC 5/5/5 vaccine containing 2.5µg of each of PRP-TT, MenA-TT and MenC-TT.

The Hib-MenAC vaccine formulations were mixed extemporaneously with Tritanirix-HepB. GSK Biologicals' combined diphtheria-tetanus-whole cell Bordetella pertussis - hepatitis B (DTPw-HB) vaccine (Tritanrix-HepB) contains not less than 30 International Units (IU) of diphtheria toxoid, not less than 60 IU of tetanus toxoid, not less than 4IU of killed Bordetella pertussis and 10µg of recombinant hepatitis B surface antigen.

Reference therapy, dose, mode of administration, lot No.:

Vaccination schedule/site: One group received Tritanrix.-HepB vaccine intramuscularly in the left thigh and Hiberix™ intramuscularly in the right thigh at 6, 10 and 14 weeks of age. Another group received Tritanrix™-HepB/Hiberix™ vaccine intramuscularly in the left thigh and Meningitec vaccine intramuscularly in the right thigh at 6, 10 and 14 weeks of age. Vaccine/composition/dose/lot number: The Tritanrix™-HepB vaccine used was as described above. One dose (0.5 ml) of GSK Biologicals' Haemophilus influenzae type b conjugate vaccine: Hiberix™ contained 10 µg of PRP conjugated to tetanus toxoid. In the Hiberix™ Group, it was mixed with sterile diluent and in the Meningitec™ Group it was mixed with Tritanrix™-HepB. One dose (0.5 ml) of Wyeth Lederle's MENINGITEC™ vaccine contained: 10 µg of capsular oligosaccharide of meningococcal group C conjugated to 15 µg of Corynebacterium diphtheria CRM197 protein and aluminium as salts.

Results - immune responses generated against Hib, MenA and MenC

Group	2.5/2.5/2.5			2.5/5/5			5/5/5			Hiberix™			Meningitec™
	%	95%CL		%	95%CL		%	95%CL		%	95%CL		%	95%CL
	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL
%≥0.15	100	96.5	100	99.0	94.8	100	100	96.5	100	100	96.5	100	100	96.5	100
GMC	20.80	15.96	27.10	22.62	17.72	28.88	19.36	15.33	24.46	38.55	29.93	49.64	10.94	8.62	13.88

Group	2.5/2.5/2.5			2.5/5/5			5/5/5			Hiberix™			Meningitec™
	%	95%CL		%	95%CL		%	95%CL		%	95%CL		%	95%CL
	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL
%≥1:8	99	94.7	100	100	96.5	100	100	96.5	100	2.9	0.6	8.4	100	96.5	100
GMT	3132	2497	3930	4206	3409	5189	3697	3118	4384	4.7	3.9	5.6	4501	3904	5180

Group	2.5/2.5/2.5			2.5/5/5			5/5/5			Hiberix™			Meningitec™
	%	95%CL		%	95%CL		%	95%CL		%	95%CL		%	95%CL
	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL
%≥1:8	99.7	91.9	99.7	100	95.8	100	100	96.2	100	6.8	2.5	14.3	9.1	4.0	17.1
GMT	316.7	251.4	398.9	418.5	358.6	488.5	363	310.5	424.4	5.6	4.3	7.4	5.6	4.4	7.2

Group	2.5/2.5/2.5			2.5/5/5			5/5/5			Hiberix™			Meningitec™
	%	95%CL		% 95%CL			%	95%CL		%	95%CL		%	95%CL
	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL
%≥0.3	100	96.5	100	100	96.4	100	100	96.5	100	8.2	3.6	15.6	100	96.5	100
GMC	49.03	43.24	55.59	71.11	62.49	80.92	61.62	54.88	69.20	0.17	0.15	0.19	58.02	51.42	65.46

Group	2.5/2.5/2.5			2.5/5/5			5/5/5			Hiberix™			Meningitec™
	%	95%CL		%	95%CL		%	95%CL		%	95%CL		%	95%CL
	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL	GMC/T	LL	UL
%≥0.3	100	96.4	100	100	96.5	100	99.0	94.8	100	1.0	0.0	5.4	5.9	2.2	12.5
GMC	18.10	15.34	21.35	26.51	22.93	30.79	23.40	20.05	27.30	0.15	0.15	0.15	0.17	0.15	0.18

Conclusion

A comparison of the immunogenicity results achieved using the oligosaccharide MenC-CRM197 conjugate vaccine and the three GSK formulations which contain polysacharide MenA-TT and MenC -TT conjugates showed that the polysaccharide Men conjugates were able to elicit a good immunogenic response similar to that achieved using the oligosaccharide conjugate vaccine Meningitec. All formulations tested gave a response to MenC in 100% of patients.

Example 5 - Phase II clinical trial administering Hib MenCY concomitantly with Infanrix penta according to a 2, 3 and 4 month schedule

Study design: A Phase II, open (partially double-blind*) randomized controlled multi-center study with 5 groups receiving a three-dose primary schedule with vaccines as follows:

• Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5) + Infanrix™ penta
• Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10) + Infanrix™ penta
• Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5) + Infanrix™ penta
• Group Hib-MenC: Hib-MenC (5/5) + Infanrix™ penta
• Group Menjugate: Menjugate™** + Infanrix™ hexa (control). *Hib-MenCY 2.5/5/5, Hib-MenCY 5/10/10 and Hib-MenC were administered in a double-blind manner while the Hib-MenCY 5/5/5 group and the Menjugate group were open. The 2.5/5/5, 5/10/10 and 5/5/5 formulations of Hib-MenCY contain MenC native polysaccharides and MenY polysaccharides which are microfluidized.**Menjugate™ contains 10µg of MenC oligosaccharides conjugated to 12.5-25µg of CRM197 per dose and is produced by Chiron.

Vaccination at +/- 2, 3, 4 months of age (Study Month 0, Month 1 and Month 2), and blood samples (3.5ml) from all subjects prior to and one month post primary vaccination (Study Month 0 and Month 3).

Study vaccine, dose, mode of administration, lot number: Three doses injected intramuscularly at one month intervals, at approximately 2, 3 and 4 months of age as follows: Table 6: Vaccines administered (study and control), group, schedule/site and dose

Hib-MenCY 2.5/5/5	2, 3, and 4	Hib (2.5µg)-MenC-TT (5µg)-MenY-TT (5 µg)	DTPa-HBV-IPV (Infanrix™ penta)
Hib-MenCY 5/10/10	2, 3, and 4	Hib (5µg)-MenC-TT (10µg)-MenY-TT (10µg)	DTPa-HBV-IPV (Infanrix™ penta)
Hib-MenCY 5/5/5	2, 3, and 4	Hib (5µg)-MenC-TT ( 5µg)-MenY-TT (5µg)	DTPa-HBV-IPV (Infanrix™ penta)
Hib-MenC	2, 3, and 4	Hib (5µg)-Men C (5µg)	DTPa-HBV-IPV (Infanrix™ penta)
Menjugate™	2, 3, and 4	Menjugate™	DTPa-HBV-IPV/Hib (Infanrix™hexa)

Immunogenicity: Measurement of antibody titres/concentrations against each vaccine antigen: Prior to the first dose (Month 0) and approximately one month after the third dose (Month 3) in all subjects for: SBA-MenC and SBA-MenY, anti-PSC and anti-PSY, anti-PRP, anti-T, anti-FHA, anti-PRN and anti-PT. Using serum bactericidal activity against N. meningitidis serogroups C and Y (SBA-MenC and SBA-MenY cut-off: 1:8 and 1:128); ELISA assays with cut-offs: ≥0.3 µg/ml and ≥2µg/ml for anti- N. meningitidis serogroups C and Y polysaccharides (anti-PSC IgG and anti-PSY IgG); ≥0.15 µg/ml and ≥1.0µg/ml for Hib polysaccharide polyribosil-ribitol-phosphate (anti-PRP IgG); 5EL.U/ml for anti-FHA, anti-PRN, anti-PT; ≥0.1 IU/ml anti-tetanus toxoid (anti-TT). Only at one month after the third dose (Month 3) in all subjects for: anti-D, anti-HBs and anti-polio 1, 2 and 3. Using ELISA assays with cut-offs: 0.1 IU/ml for anti-diphtheria (anti-D); ≥10 mlU/ml for antihepatitis B (anti-HBs); and microneutralization test cut-off: 1:8 for anti-polio type 1, 2 and 3 (anti-polio 1, 2 and 3).

Statistical methods:

The seroprotection/seropositivity rates and geometric mean concentrations/titres (GMCs/GMTs) with 95% confidence intervals (95% CI) were computed per group, for SBA-MenC, anti-PSC, SBA-MenY, anti-PSY, anti-PRP, anti-Tetanus, anti-PT, anti-FHA and anti-PRN prior to and one month after vaccination; for anti-Diphtheria, anti-HBs, anti-Polio 1, anti-Polio 2 and anti-Polio 3 one month after vaccination. Vaccine response (appearance of antibodies in subjects initially seronegative or at least maintenance of antibody concentrations in subjects initially seropositive) with 95% Cl for anti-PT, anti-PRN and anti-FHA were also computed one month after vaccination. Reverse cumulative curves for each antibody at Month 3 are also presented. The differences between the Hib-MenCY and the Hib- MenC groups, compared with the Menjugate™ control group were evaluated in an exploratory manner for each antibody, except for SBA-MenY and anti-PSY, in terms of (1) the difference between the Menjugate™ group (minus) the Hib-MenCY and Hib-MenC groups for the percentage of subjects above the specified cut-offs or with a vaccine response with their standardized asymptotic 95% CI, (2) the GMC or GMT ratios of the Menjugate™ group over the Hib-MenCY and Hib-MenC groups with their 95% CI. The same comparisons were done to evaluate the difference between each pair of Hib-MenCY formulations for anti-PRP, SBA-MenC, anti-PSC, SBA-MenY, anti-PSY and anti-TT antibodies.

Seroprotection/seropositivity rates &GMC/Ts (ATP cohort for immunogenicity)

Group	N	%≥0.15	LL	UL	≥1	LL	UL	GMC	LL	UL
Hib MenCY 2.5/5/5	67	100.0	94.6	100.0	98.5	92.0	100.0	9.01	7.25	11.21
Hib MenCY 5/10/10	67	100.0	94.6	100.0	98.5	92.0	100.0	9.49	7.72	11.65
Hib MenCY 5/5/5	70	100.0	94.9	100.0	98.6	92.3	100.0	8.08	6.53	9.98
Hib MenC	74	100.0	95.1	100.0	98.6	92.7	100.0	10.44	8.49	12.83
Menjugate™	71	100.0	94.9	100.0	80.3	69.1	88.8	2.60	1.97	3.43

Group	N	%≥ 1:8	LL	UL	≥1:128	LL	UL	GMT	LL	UL
Hib MenCY 2.5/5/5	70	100.0	94.9	100.0	95.7	88.0	99.1	1005.8	773.5	1308.0
Hib MenCY 5/10/10	67	100.0	94.6	100.0	94.0	85.4	98.3	1029.8	799.7	1326.0
Hib MenCY 5/5/5	71	100.0	94.9	100.0	94.4	86.2	98.4	906.9	691.3	1189.8
Hib MenC	74	100.0	95.1	100.0	95.9	88.6	99.2	871.0	677.3	1120.0
Menjugate™	71	100.0	94.9	100.0	100.0	94.9	100.0	3557.6	2978.8	4248.8

Group	N	%≥0.3	LL	UL	≥2	LL	UL	GMC	LL	UL
Hib MenCY 2.5/5/5	69	100.0	94.8	100.0	100.0	94.8	100.0	21.70	18.36	25.65
Hib MenCY 5/10/10	66	100.0	94.6	100.0	100.0	94.6	100.0	27.26	23.26	31.95
Hib MenCY 5/5/5	70	100.0	94.9	100.0	100.0	94.9	100.0	19.02	16.49	21.93
Hib MenC	74	100.0	95.1	100.0	100.0	95.1	100.0	21.08	18.24	24.35
Menjugate™	71	100.0	94.9	100.0	100.0	94.9	100.0	38.49	33.64	44.05

Group	N	%≥ 1:8	LL	UL	≥1:128	LL	UL	GMT	LL	UL
Hib MenCY 2.5/5/5	69	97.1	89.9	99.6	92.8	83.9	97.6	470.7	351.1	631.2
Hib MenCY 5/10/10	66	97.0	89.5	99.6	86.4	75.7	93.6	437.1	322.0	593.4.8
Hib MenCY 5/5/5	71	98.6	92.4	100.0	95.8	88.1	99.1	635.3	501.5	804.8
Hib MenC	74	21.6	12.9	32.7	13.5	6.7	23.5	9.3	6.3	13.7
Menjugate™	71	19.7	11.2	30.9	9.9	4.1	19.3	7.5	5.4	10.4

Group	N	%≥0.3	LL	UL	≥2	LL	UL	GMC	LL	UL
Hib MenCY 2.5/5/5	69	100.0	94.8	100.0	100.0	94.8	100.0	26.86	22.86	31.56
Hib MenCY 5/10/10	66	100.0	94.6	100.0	100.0	94.6	100.0	37.02	31.84	43.04
Hib MenCY 5/5/5	70	100.0	94.9	100.0	100.0	94.9	100.0	23.57	19.94	27.86
Hib MenC	74	8.1	3.0	16.8	4.1	0.8	11.4	0.19	0.15	0.25
Menjugate ™	71	5.6	1.6	13.8	1.4	0.0	7.6	0.17	0.15	0.19

Group	N	%≥ 0.1	LL	UL	GMC	LL	UL
Hib MenCY 2.5/5/5	68	100.0	94.7	100.0	3.06	2.63	3.55
Hib MenCY 5/10/10	67	100.0	94.6	100.0	3.25	2.88	3.68
Hib MenCY 5/5/5	70	100.0	94.9	100.0	2.97	2.59	3.41
Hib MenC	74	100.0	95.1	100.0	3.15	2.73	3.64
Menjugate™	71	100.0	94.9	100.0	1.66	1.39	1.97

Conclusion

The MenC and Y polysaccharide conjugates produced a good immune response in all subjects with 100% of subjects producing above 0.3 µg/ml responses against MenC and MenY.

Example 6 - Phase II clinical trial comparing three formulations of MenACWY-TT with Meningitec MenC-CRM197 oligosaccharide-conjugate vaccine.

This example reports a phase II, open (partially-blind), randomized, controlled dose-range study to evaluate the Immunogenicity of three different formulations of GlaxoSmithKline Biological's meningococcal serogroups A, C, W-135, Y tetanus toxoid conjugate (MenACWY-TT) vaccine in comparison to a MenC oligosaccharide-CRM197 conjugate vaccine (Meningitec) when given as one dose to children aged 12-14 months.

The clinical trial was an open (partially double-blind*), controlled, multicentric study in which eligible subjects of 12-14 months were randomized (1:1:1:1) to one of four parallel groups of 50 subjects to receive a single primary dose at Visit 1 as follows:

• Form 1T: MenACWY-TT at a dose of 2.5µg of MenA polysaccharide conjugated to tetanus toxoid (TT), 2.5µg of MenC polysaccharide conjugated to TT, 2.5µg of MenW polysaccharide conjugated to TT and 2.5µg of MenY polysaccharide conjugated to TT.
• Form 2T: MenACWY-TT at a dose of 5µg of MenA polysaccharide conjugated to TT, 5µg of MenC polysaccharide conjugated to TT, 5µg of MenW polysaccharide conjugated to TT and 5µg of MenY polysaccharide conjugated to TT.
• Form 3T: MenACWY-TT at a dose of 2.5µg of MenA polysaccharide conjugated to TT, 10µg of MenC polysaccharide conjugated to TT, 2.5µg of MenW polysaccharide conjugated to TT and 2.5µg of MenY polysaccharide conjugated to TT.
• Ctrl T: 10µg MenC oligosaccharide conjugated to 12.5-25µg CRM197 (Meningitec™). *The three different MenACWY-TT formulations were administered in a double-blind manner.

Vaccination schedule/site: A single vaccine dose was administered intramuscularly in the left deltoid at Visit 1 (Study Month 0) according to randomized assignment. All candidate vaccines were supplied as a lyophilized pellet in a monodose vial (0.5 ml after reconstitution with the supplied saline diluent).

Immunogenicity: Measurement of titers/concentrations of antibodies against meningococcal vaccine antigen components in blood samples obtained prior to the study vaccine dose (Month 0) and approximately one month after the study vaccine dose (Month 1) in all subjects. Determination of bactericidal antibody titers against N. meningitidis serogroups A, C, W-135 and Y (SBA-MenA, SBA-MenC, SBA-MenW and SBA-MenY) by a bactericidal test (assay cut-offs: a dilution of 1:8 and 1:128) and ELISA measurement of antibodies against N. meningitidis serogroups A, C, W-135 and Y (anti-PSA, anti-PSC, anti-PSW and anti-PSY, assay cut-offs ≥0.3µg/ml and ≥2µg/ml), and tetanus toxoid (anti-tetanus, assay cut-off 0.1 IU/ml).

Results

Antibody response in terms of the percentage of SBA-MenA, SBA-MenC, SBA-MenW and SBA-MenY responders one month after vaccination (the primary endpoint) is shown in Table 8. A response is defined as greater than or equal to a 4-fold increase for seropositive subjects or seroconversion for seronegative subjects before vaccination.

Antibody	Group	N	%	LL	UL
SBA-MenA	Form 1T	42	61.9	45.6	76.4
Form 2T	39	82.1	66.5	92.5
Form 3T	40	62.5	45.8	77.3
Meningitec™	36	11.1	3.1	26.1
SBA-MenC	Form 1T	46	97.8	88.5	99.9
Form 2T	43	100.0	91.8	100.0
Form 3T	44	95.5	84.5	99.4
Meningitec™	49	91.8	80.4	97.7
SBA-MenW	Form 1T	45	100.0	92.1	100.0
Form 2T	43	97.7	87.7	99.9
Form 3T	45	100.0	92.1	100.0
Meningitec™	46	15.2	6.3	28.9
SBA-MenY	Form 1T	47	97.9	88.7	99.9
Form 2T	44	88.6	75.4	96.2
Form 3T	45	93.3	81.7	98.6
Meningitec™	49	4.1	0.5	14.0

Table 9 shows the numbers of subjects achieving SBA titres over cutoff points of 1:8 and 1:128 as well as GMTs.

	Group	N	%	≥1:8 LL	UL	%	≥1:128 LL	UL	GMT
SBA-MenA	Form 1T	46	100	92.3	100	100	92.3	100	1457.3
	Form2T	45	100	92.1	100	97.8	88.2	99.9	1776.9
	Form3T	48	97.9	88.9	99.9	97.9	88.9	99.9	1339.5
	Meningitec™	41	51.2	35.1	67.1	43.9	28.5	60.3	42.8
SBA-MenC	Form 1T	47	97.9	88.7	99.9	78.7	64.3	89.3	281.3
	Form2T	45	100	92.1	100	84.4	70.5	93.5	428.6
	Form3T	47	95.7	85.5	99.5	85.1	71.7	93.8	478.4
	Meningitec™	50	94.0	83.5	98.7	62.0	47.2	75.3	200.1
SBA-Men W	Form 1T	47	100	92.5	100	100	92.5	100	2529.1
	Form2T	45	100	92.1	100	100	92.1	100	2501.6
	Form3T	48	100	92.6	100	97.9	88.9	99.9	2300.2
	Meningitec™	48	27.1	15.3	41.8	6.3	1.3	17.2	9.4
SBA-MenY	Form 1T	47	100	92.5	100	100	92.5	100	1987.4
	Form2T	45	100	92.1	100	100	92.1	100	2464.8
	Form3T	48	100	92.6	100	97.9	88.9	99.9	2033.7
	Meningitec™	49	49.0	34.4	63.7	28.6	16.6	43.3	25.0

Vaccination with all three formulations of the ACWY-TT polysaccharide conjugate led to good SBA responses against MenA, MenC, MenW and MenY with 95-100% of subjects with titres greater than 1:8. In particular, the 5/5/5/5 and 2.5/10/2.5/2.5 formulations of the polysaccharide conjugates produced a higher response against MenC than the oligosaccharide Meningitic vaccine as seen by a higher proportion of subjects having a titre greater than 1:128 and the GMT readings.

	Group	N	%	≥0.3µg /ml LL	UL	%	≥2µg/ ml LL	UL	GMC µg/ml
Anti-MenA	Form 1T	47	93.6	82.5	98.7	68.1	52.9	80.9	2.35
	Form2T	45	100	92.1	100	64.4	48.8	78.1	3.11
	Form3T	48	95.8	85.7	99.5	37.5	24.0	52.6	1.65
	Meningitec™	50	10.0	3.3	21.8	2.0	0.1	10.6	0.18
Anti-MenC	Form 1T	47	100	92.5	100	100	92.5	100	9.57
	Form2T	45	100	92.1	100	100	92.1	100	12.53
	Form3T	47	100	92.5	100	97.9	88.7	99.9	19.29
	Meningitec™	49	98.0	89.1	99.9	93.9	83.1	98.7	7.95
Anti-Men W	Form 1T	47	100	92.5	100	80.9	66.7	90.9	4.56
	Form2T	45	100	92.1	100	93.3	81.7	98.6	6.83
	Form3T	48	93.8	82.8	98.7	72.9	58.2	84.7	2.88
	Meningitec™	50	0.0	0.0	7.1	0.0	0.0	7.1	0.15
Anti-MenY	Form 1T	47	100	92.5	100	97.9	88.7	99.9	8.90
	Form2T	45	100	92.1	100	100	92.1	100	12.78
	Form3T	47	97.9	88.7	99.9	87.2	74.3	95.2	5.67
	Meningitec™	50	2.0	0.1	10.6	0.0	0.0	7.1	0.15

All three formulations of the ACWY-TT polysaccharide conjugate vaccine produced good immune responses against MenA, MenC, MenW and MenY with between 93% and 100% of subjects achieving titres grater than 0.3µg/ml. Higher GMC readings were achieved using the 5/5/5/5 and 2/5/10/2.5/2.5 formulations of the ACWY-TT polysaccharide conjugate vaccine in comparison with Meningitec™.

Example 7 - comparison of immunogenicity of native and sized MenY polysaccharide conjugates

Mice (female DBA/2 of 6-8 wk) received two injections, 2 weeks apart, of PSY-TT by the subcutaneous route. Blood samples were taken 14 days after the second injection in order to perform anti-PSY ELISA and SBA using S1975 menY strain. Per injection, mice received 1 µg of PSY-TT(lyo non-ads formulation).

The conjugates described in table 11 were used . Table 11

Conjugates	ENYTT012	ENYTT014	ENYTT015 bis
PSY microfluidisation	NO	Yes (40 cycles)	Yes (20 cycles)
TT/PS ratio	1/1	1/1	1/1

Results

The results (Figure 1) show a trend towards higher immunogenicity for conjugates prepared using sized PSY. Figure 1A shows the GMC results obtained in an ELISA for antisera raised against conjugates prepared from native MenY (ENYTT012), microfluidised MenY - 40 cycles (ENYTT014) and microfluidised MenY - 20 cycles (ENYTT015 bis). Higher GMCs were obtained where the MenY-TT was prepared from microfluidised MenY.

Similar results were obtained when the antisera were assessed by SBA assay (Figure 1B). Again the higher GMT values were achieved using conjugates prepared from microfluidised MenY.

Claims (14)

1. An immunogenic composition that either does not use any aluminium salt adjuvant or does not use any adjuvant at all, the composition comprising N. meningitidis capsular polysaccharides from at least one of serogroups A, C, W135 and Y conjugated to a carrier protein to produce a N. meningitidis capsular polysaccharide conjugate, wherein the average size of each N. meningitidis polysaccharide is above 50kDa, wherein each N. meningitidis polysaccharide is conjugated to tetanus toxoid carrier and is either a native polysaccharide or is sized by microfluidization, wherein said composition comprises a MenC capsular polysaccharide conjugated to tetanus toxoid and said MenC capsular polysaccharide has an average size of 150-210 kDa.
2. The immunogenic composition of claim 1 further comprising a MenA capsular polysaccharide conjugated to tetanus toxoid.
3. The immunogenic composition of any one of claims 1-2, wherein said MenC capsular polysaccharide has an average size of 150-180 kDa.
4. The immunogenic composition of any one of claims 1-3 further comprising a MenY capsular polysaccharide conjugated to tetanus toxoid.
5. The immunogenic composition of any one of claims 1-4 further comprising a MenW capsular polysaccharide conjugated to tetanus toxoid.
6. The immunogenic composition of any preceding claim wherein each N. meningitidis capsular polysaccharide conjugate has a polysaccharide:carrier ratio of 1:5-5:1 or 1:1-1:4(w/w).
7. The immunogenic composition of any preceding claim further comprising a H. influenzae b capsular saccharide conjugated to a carrier protein.
8. The immunogenic composition of claim 7 wherein the H. influenzae b capsular saccharide is conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197, fragment C of TT and protein D.
9. The immunogenic composition of claim 7 or 8 wherein the ratio of Hib to carrier protein in the Hib capsular saccharide conjugate is between 1:5 and 5:1 (w/w) or between 1:1 and 1:4, 1:2 and 1:3.5 or around 1:3 (w/w).
10. The immunogenic composition of any one of claims 1-9 comprising a N. meningitidis serogroup B outer membrane vesicle preparation or capsular saccharide.
11. A vaccine comprising the immunogenic composition of any one of claims 1-10 and a pharmaceutically acceptable carrier.
12. A vaccine kit for concomitant or sequential administration comprising two multi-valent immunogenic compositions for conferring protection in a host against disease caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Haemophilus influenzae and Neisseria meningitidis, said kit comprising a first container comprising :

    tetanus toxoid (TT),

    diphtheria toxoid (DT), and

    wholecell or acellular pertussis components

    and a second container comprising: the immunogenic composition of any one of claims 1-10.
13. A process for making the vaccine of claim 11 comprising the step of mixing the immunogenic composition of any one of claims 1-10 with a pharmaceutically acceptable carrier.
14. The immunogenic composition of claims 1-10 for use in the treatment or prevention of disease caused by Neisseria meningitidis infection.