MX2007007686A - Saccharide conjugate vaccines - Google Patents

Abstract

The invention provides compositions comprising a combination of two or more monovalent conjugates, each of said two or more monovalent conjugates comprising a carrier protein comprising T cell epitopes from two or more pathogens conjugated to saccharide antigen. The invention also provides a multivalent conjugate comprising two or more antigenically distinct saccharide antigens conjugated to the same carrier protein molecule, wherein the carrier protein comprises T cell epitopes from two or more pathogens. Further compositions comprise one or more of said monovalent conjugates and one or more of said multivalent conjugates. The invention further provides methods for making said compositions and uses for said compositions. The examples involve the conjugation of Nl 9 carrier protein to meningococcal oligosaccharides.

Description

VACCINES OF CONJUGADOS SACARIDOS FIELD OF THE INVENTION This invention is in the field of vaccines and refers to new compositions comprising two or more saccharide antigens conjugated to a polyepitopic carrier protein comprising T cell epitopes from multiple pathogenic proteins. The invention also relates to the methods for making said compositions and to the uses for the compositions.

BACKGROUND OF THE INVENTION Multivalent vaccines are known in the art. One such example is a tetravalent vaccine of capsular polysaccharides from serogroups A, C, Y and 135 of N. meningitidis, which has been known for many years [1, 2] and has been authorized for use in humans. However, although it is effective in adolescents and adults, it induces a poor immune response and short duration of protection and can not be used in infants [for example, 3]. This is because the polysaccharides are antigens independent of T cells that induce in general a weak immune response that can not be reinforced. Interests have been emerging regarding the widespread use of polyvalent vaccines because they are subject to a REF. : 183485 significant decrease in immune function known as immunosuppression. Immunosuppression can result when the amount of antigen introduced into the subject exceeds the ability of the immune system to respond. Such a condition is referred to as an antigen load. Immunosuppression can also occur as a result of an antigenic component that prevents the immune system from responding to another antigenic component of a polyvalent vaccine. This last form of immunosuppression is called vaccine interference. In the last 20 years, conjugate vaccines have been developed, comprising bacterial capsular polysaccharides conjugated to carrier proteins. Examples include the conjugate vaccine of Haemophilus influenzae type b (Hib) [4] as well as the conjugate vaccines against Streptococcus pneumoniae [5] and Neisseria miningitidis of serogroup C (MenC) [6]. Carrier proteins used in licensed vaccines include tetanus toxoid (TT), diphtheria toxoid (DT), non-toxic diphtheria toxin mutant CRM197, and the outer membrane protein complex of N. meningitidis. group B. Since more conjugate vaccines are being introduced into medical practice, infants may receive multiple injections of the carrier protein, either as a vaccine itself (for example, TT or DT) or as a carrier protein present in a conjugate vaccine. Since these proteins are highly immunogenic at the level of B cells and T cells, carrier overload can induce immune suppression in primed or primed individuals [7]. This phenomenon, called carrier-induced epitope suppression, is thought to be due to carrier-specific antibodies and intramolecular antigenic competition [8]. Ideally, a carrier protein should induce strong helper or helper effect for a conjugated B cell epitope (e.g., polysaccharide) without inducing an antibody response against itself. The use of universal epitopes, which are immunogenic in the context of most of the class II molecules of the major histocompatibility complex, is an approach to a certain goal [9]. Such epitopes have been identified within TT and other proteins. However, there is still a need for additional improvements. It is therefore an object of the invention to provide improved saccharide conjugates.

BRIEF DESCRIPTION OF THE INVENTION It has been found that polyepitopic carrier proteins are particularly useful as carriers for saccharide combinations. In addition, it has been discovered that only a low immunogenic response against these carrier proteins is observed, even when they comprise a number of known pathogenic epitopes, whereas it would have been expected that the immunogenic response could increase proportionally to the number of pathogenic epitopes. In some embodiments, the invention therefore provides a composition comprising a combination of two or more monovalent conjugates (eg, 2, 3, 4, 5, 6, or more, see Figure 1A). Each monovalent conjugate comprises (i) a carrier protein comprising T cell epitopes derived from two or more (e.g., 2, 3, 4, 5, 6, or more) pathogens conjugated to (ii) a saccharide antigen. Preferably, the carrier protein used in each conjugate is the same, preferably, at least one of the epitopes of the carrier protein is not derived from the same pathogen as the saccharide antigen. Preferably, none of the carrier protein epitopes is derived from the same pathogen as the saccharide antigen. Although each carrier protein molecule in each monovalent conjugate can be conjugated to more than one saccharide antigen molecule (eg, 1, 5, 10, 20 or more) due to the multiple binding sites on each carrier protein molecule (FIG. IB), each saccharide antigen conjugated to any given carrier protein, is preferably from the same antigenically distinct pathogen. For example, saccharide antigens from MenA are derived from those from each of MenC, MenW and enY and are therefore said to be derived from antigenically distinct pathogens, while saccharide antigens from Hib are all from the same pathogen antigenically different. In a simple conjugate, the individual saccharides, although they come from the same antigenically distinct pathogen, can be of different chain lengths. As an alternative, in some embodiments, the invention provides a multivalent conjugate comprising two or more (e.g., 2, 3, 4, 5, 6 or more) antigenically distinct antigens, conjugated to the same carrier protein molecule (FIG. 1 C) . In this case, the saccharide antigens are derived from different antigenically distinct pathogens. Thus, for example, in such conjugated composition, each carrier protein molecule can have saccharide antigens from two or more of MenA, MenC, MenW, MenY and Hib conjugated to it. The invention also provides a composition comprising two or more (eg, 2, 3, 4, 5, 6 or more) of those conjugates. As a further alternative, the invention provides a composition comprising one or more (by example, 1, 2, 3, 4, 5, 6 or more) monovalent conjugates and one or more (eg, 1, 2, 3, 4, 5, 6 or more) multivalent conjugates as described above.

Carrier protein Carrier protein may comprise 2 or more epitopes of T cells (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or plus) . Preferably, the carrier protein comprises 6 or more, or 10 or more epitopes. More preferably, the carrier protein comprises 19 or more epitopes. Each carrier protein may only have one copy of a particular epitope or may have more than one copy of a particular epitope. Preferably, the epitopes are epitopes of CD4 + T cells. Preferably, the carrier protein comprises at least one bacterial epitope and at least one viral epitope. Preferably, the epitopes are derivatives of antigens to which the human population is frequently exposed, either by natural infection or by vaccination, for example, the epitopes can be derived from the hepatitis A virus, hepatitis B virus, measles virus , influenza virus, varicella zoster virus, heat shock proteins from microbacterium bovis and strains of M. leprae and / or Streptococcus, etc. Preferably, the epitopes are selected from tetanus toxin (TT), Plasmodium falciparum CSP (PfCs), hepatitis B virus core capsid (HBVnc), influenza inina hemagglut (HA), surface HBV antigen ( HBsAg), and influenza (MT) matrix. The epitopes used in the carrier protein are preferably selected from P23TT (SEQ ID No. 1), P32TT (SEQ ID No. 2), P21TT (SEQ ID No. 3), PfCs (SEQ ID No. 4) , P30TT (SEQ ID No.: 5), P2TT (SEQ ID No.:6), HBVnc (SEQ ID No.: 7), HA (SEQ ID No.: 8), HBsAg (SEQ ID No.:9) and MT (SEQ ID No.: 10). Preferably, the epitopes are joined by spacers. Preferably, the spacer is a short sequence of amino acids (eg, 1, 2, 3, 4 or 5) which is not an epitope. A preferred spacer comprises one or more glycine residues, for example, -KG-. Preferably, the carrier protein comprises a N- or C-terminal region comprising a six-His tail, an immunoaffinity marker useful for selecting the carrier protein (e.g., the sequence "MDYKDDDD" [SEQ ID NO: 12] may be used) and / or a protease cleavage sequence. Preferably, the proteolytic sequence is the factor Xa recognition site. Preferably, the carrier does not comprise suppressor T cell epitopes.

Preferably, the carrier protein is N19 (SEQ ID No .: 11). It has been shown that a genetically engineered protein, called N19 [10], expressed in Escherichia coli and having several universal epitopes of human CD4 + T cells, behaves like a strong carrier when conjugated to the polysaccharide Hib [11]. The N-terminal region of N19 consists of (i) a tail of six His that can be exploited during purification, (ii) a marker peptide sequence (Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp (SEQ ID No .: 12) recognized by a rabbit polyclonal antibody that can be used to select positive colonies during the cloning procedure, (iii) the factor Xa recognition site Ile-Glu-Gly-Arg (SEQ ID No .: 13) for the easy elimination of the marker N19 is a duplication of the first nine epitopes listed in Table 1, plus the epitope MT of CD4 + influenza matrix.The epitopes are separated by a Lys-Gly spacer to provide flexibility to the molecule and to allow subsequent conjugation of the polysaccharide to the primary e-amino groups of the Lys residues In addition to the CD4 + epitopes, the carrier proteins may comprise other peptides or protein fragments, such as epitopes from the immune cytokines. omodulators such as interleukin 2 (IL-2) or the granulocyte-macrophage colony stimulation factor (G -CSF).

Table 1 Saccharide antigens Preferably, the saccharide antigen conjugated to the carrier protein in a composition of the invention is a bacterial saccharide and in particular a bacterial capsular saccharide. Examples of bacterial capsular saccharides that can be included in the compositions of the invention include capsular saccharides of Neisseria meningitidis (serogroups A, B, C, W135 and / or Y), Streptococcus pneumoniae (serotypes 1, 2, 3, 4, 5 , 6B, 7F, 8, 9N, 9V, 10A, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, particularly 4, 6B, 9V, 14, 18C, 19F and / or 23F), Streptococcus agalactiae (types Ia, Ib, II, III, IV, V, VI, VII, and / or VIII, such as the saccharide antigens described in references 20-23), Haemophilus influenzae (typable strains: a, b, c, d, e and / of), Pseudomonas aeruginosa (for example LPS isolated from PA01, serotype 05, Staphylococcus aureus (from, for example, serotypes 5 and 8), Enterococcus faecalis or E.faecium (repetitions of t laughtercarids), Yersinia enterocolitica, Vibrio cholerae, Salmonella typhi, Klebsiella spp., etc. Other saccharides that may be included in the compositions of the invention include glucans (for example, plo, fungal glycans, such as those of Candida albicans), and fungal capsular saccharides, for example from the capsule of Cryptococcus neoformans. The capsule of N. meningitidis serogroup A (MenA) is a homopolymer of N-acetyl-D-mannosamine-1-phosphate (al-> 6) -linked, with partial O-acetylation at the C3 positions and C4 The capsule of N. meningitidis serogroup B (MenB) is a sialic acid homopolymer (a2- »8) -linked. The capsular saccharide of N. meningitidis serogroup C (MenC) is a homopolymer of sialic acid (a2- »9) -linked, with variable 0-acetylation at positions 7 and / or 8. The saccharide of serogroup W135 from N. meningitidis is a polymer that has disaccharides of sialic acid-galactose [- »4) -D-Neup5Ac (7 / 90Ac) -a- (2? 6) -D-Gal-a- (1?). This has the 0 -acetylation variable in positions 7 and 9 of sialic acid [24] The saccharide of serogroup Y of N. meningitidis is similar to the saccharide of serogroup W135, except that the repeated disaccharide unit includes glucose instead of galactose [? 4) - D-Neup5Ac (7 / 90Ac) -a- (2? 6) -D-Glc- - (1?).

It also has variable 0-acetylation at positions 7 and 9 of the sialic acid. The compositions of the invention comprise mixtures of saccharide antigens. Preferably, the compositions comprise 2, 3, 4 or more different saccharide antigens. The antigens may be derived from the same or from antigenically distinct pathogens. Preferably, the compositions of the invention comprise saccharide antigens from more than one serogroup of N. meningitidis, for example, the compositions may comprise conjugates of saccharides from serogroups A + C, A + 135, A + Y, C + 135, C + Y, 135 + Y, A + C + 135, A + C + Y, C + 135 + Y, A + C + W135 + Y, etc. Preferred compositions comprise saccharides of serogroups C and Y. Other preferred compositions comprise saccharides of serogroups C, W135, and Y. Particularly preferred compositions comprise saccharides of serogroups A, C, W135 and Y. Where a mixture comprises meningococcal saccharides. of serogroup A and at least one other serogroup saccharide, the ratio (weight / weight) of the MenA saccharide to any other serogroup saccharide may be greater than 1 (eg, 2: 1, 3: 1, 4: 1, 5: 1, 10: 1 or greater). The proportions between 1: 2 and 5: 1 are preferred, as are the proportions between 1: 1.25 and 1: 2.5. The preferred proportions (weight / weight) for the saccharides of serogroups A: C: W135: Y are: 1: 1: 1: 1; 1: 1: 1: 2; 2: 1: 1: 1; 4: 2: 1: 1; 8: 4: 2: 1; 4: 2: 1: 2; 8: 4: 1: 2; 4: 2: 2: 1; 2: 2: 1: 1; 4: 4: 2: 1; 2: 2: 1: 2; 4: 4: 1: 2; and 2: 2: 2: 1. Additional compositions of the invention comprise the conjugate of the saccharide Hib and a saccharide conjugate of at least one serogroup of N. meningitidis, preferably of more than one serogroup of N. meningitidis. For example, a composition of the invention may comprise a Hib saccharide and saccharides of one or more (eg, 1, 2, 3 or 4) of the serogroups of N. meningitidis A, C, W135 and Y. Other combinations of conjugates saccharides from the pathogens mentioned above, are also provided. The invention also provides, in some embodiments, combinations of conjugates wherein the carrier protein is not the same for each conjugate. Additional preferred compositions of the invention comprise a first conjugate and a second conjugate. The first conjugate is a polyepitopic conjugate as described above and the second conjugate comprises a saccharide antigen conjugated to a carrier protein different from that used in the first conjugate. For example, the second conjugate may be a conjugated saccharide antigen to the CRM197 carrier. The saccharide antigen (s) in the second conjugate can be the same as or different from the saccharide antigen (s) in the first conjugate.

Preparation of capsular saccharide antigens Methods for the preparation of capsular saccharide antigens are well known. For example, reference 25 describes the preparation of saccharide antigens of N. meningitidis. The preparation of saccharide antigens of H. influenzae is described in chapter 14 of ref. 26. The preparation of the conjugated saccharide antigens of S. pneumoniae is described in the art. For example, Prevenar ™ is a heptavalent pneumococcal conjugate vaccine. The Processes for the preparation of saccharide antigens of S. agalactiae are described in detail in reference 27 and 28. Capsular saccharides can be purified by known techniques, as described in several references herein. The saccharide antigens can be chemically modified. For example, these can be modified to replace one or more hydroxyl groups with blocking groups. This is particularly useful for serogroup A meningococci where acetyl groups can be replaced with blocking groups to prevent hydrolysis [29]. Such modified saccharides are still saccharides of serogroup A within the meaning of the present invention. The saccharide can be chemically modified relative to the capsular saccharide as it is found in nature. For example, the saccharide can be de-O-acetylated (partially or completely), des-N-acetylated (partially or completely), N-propionated (partially or completely), etc. Deacetylation can occur before, during, or after conjugation, but preferably occurs before conjugation. Depending on the particular saccharide, deacetylation may or may not affect immunogenicity, for example, the NeisVac-CMR vaccine uses a de-0-acetylated saccharide, while Menj ugate ™ is acetylated, but both vaccines are effective. The effect of de-acetylation, etc., can be evaluated through routine tests. Routine saccharides can be used in the form of oligosaccharides. These are conveniently formed by fragmentation of the purified capsular polysaccharide (e.g., by hydrolysis) which will usually be followed by purification in fragments of the desired size. The fragmentation of the polysaccharides is preferably performed to give a final average degree of polymerization (DP) in the oligosaccharide of less than 30. The DP can be conveniently measured by ion exchange chromatography or by colorimetric assays [30]. If hydrolysis is performed, the hydrolyzate will generally be of adequate size in order to eliminate the short-length oligosaccharides [31]. This can be achieved in various ways, such as ultrafiltration, followed by ion exchange chromatography. Oligosaccharides with a degree of polymerization of less than or equal to about 6 are preferably eliminated from meningococci of serogroup A, and those less than about 4 are preferably eliminated for serogroups W135 and Y.

Carrier-Saccharide Conjugates The conjugates of the invention may include small amounts of free carrier (eg, unconjugated). When a given carrier protein is present in free and conjugated form in a composition of the invention, the unconjugated form is preferably not greater than 5% of the total amount of the carrier protein in the composition as a whole, and more preferably present at less than 2% (by weight). After conjugation, the free and conjugated saccharides can be separated. There are many suitable methods, including hydrophobic chromatography, tangential ultrafiltration, dialfiltration, etc. [see also references 32 and 33, etc.]. Any suitable conjugation action can be used, with any suitable linker where necessary. The binding of the saccharide antigen to the carrier is preferably via a -NH2 group, for example, in the side chain of a lysine residue in a carrier protein, or of an arginine residue. Where a saccharide has a free aldehyde group, then it can react with an amine in the carrier to form a conjugate by reductive amination. The linkage can also be via a -SH group for example, in the side chain of a cysteine residue. Alternatively, the saccharide antigen can be bound to carrier via a linker molecule. The saccharide will typically be activated or functionalized before conjugation. Activation may involve, for example, cyanlation reagents such as CDPA (eg, l-cyano-4-dimethylaminopyridinium tetrafluoroborate [34, 35, etc.]). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see also introduction to reference 36).

Linkers Linkages via a linker group can be performed using any known method, for example, the procedures described in references 37 and 38. One type of linkage involves the reductive amination of the saccharide, the coupling of the resulting amino group by one end of a linker. adipid acid linker group, and then coupling the carrier protein to the other end of the adipid acid linker group [39, 40]. Other linkers include b-propionamido [41], nitrophenyl-ethylamine [42], haloacyl halides [43], glycosidic linkages [44], 6-aminocaproic acid [45], ADH [46], C4 to C12 portions [47] etc. As an alternative to the use of a linker, the direct link can be used. Direct links to protein they may comprise the oxidation of the polysaccharide, followed by the reductive amination with the protein, as described for example in references 48 and 49. A process involving the introduction of amino groups into the saccharide is preferred (for example, by replacing group = 0 terminals with -NH2) followed by derivatization with an adipic diester (for example, N-hydroxysuccinimide diester of adipic acid) and the reaction with the carrier protein. A bifunctional linker can be used to provide a first group for coupling for an amine group in the saccharide and a second group for coupling to the carrier (typically for coupling to an amine in the carrier). The first group in the bifunctional linker is thus able to react with an amine group (-NH2) on the saccharide. This reaction will typically involve an electrophilic substitution of the hydrogen of the amine. The second group in the bifunctional linker is capable of reacting with an amine group on the carrier. This reaction will again typically involve an electrophysical substitution of the amine. Where the reactions with the saccharide and the carrier involve amines, then it is preferred to use a bifunctional linker of the formula X-L-X, where: the X groups are the themselves and can react with the amines; and where L is a linking portion in the linker. A preferred group X is N-oxysuccinimide. L preferably has the formula L'-L2-L ', where L 'is carbonyl. Preferred L2 groups are straight chain alkyls with 1 to 10 carbon atoms (for example Ci, C2, C3, C4, C5, C6, C7, C8, Cg Cio) for example, - (CH2) 4-. Other X groups are those which form esters when combined with HO-L-OH, such as norborane, p-nitrobenzoic acid or sulfo-N-hydroxysuccinimide. Additional bifunctional linkers for use with the invention include acryloyl halides (e.g., chloride) and haloacyl halides. The linker will be added in general in molar excess to the modified saccharide. After conjugation, the free and conjugated saccharides can be separated. There are many suitable methods, including hydrophobic chromatography, tangential ultrafiltration, dialfitration, etc. [see also references 50 and 51, etc.]. Where the composition of the invention includes a depolymerized saccharide, it is preferred that the depolymerization precedes the conjugation.

Additional antigens The compositions of the invention may comprise one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional antigens, such as: A. Bacterial antigens Bacterial antigens suitable for use in the invention include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles that can be isolated, purified and derived from bacteria. In addition, bacterial antigens may include bacterial lysates and formulations of inactivated bacteria. Bacterial antigens can be produced by recombinant expression. Bacterial antigens preferably include epitopes that are exposed on the surface of the bacteria during at least one stage of their life cycle. Bacterial antigens are preferably conserved across multiple serotypes. Bacterial antigens include antigens derived from one or more of the bacteria described below, as well as the examples of specific antigens identified below.

Neisseria meningitidis: Meningococcal antigens may include proteins (such as those identified in references 52-58), saccharides (including a polysaccharide, an oligosaccharide or a lipopolysaccharide), or outer membrane vesicles [59-62] purified or derived from a serogroup of N. meningitidis such as A, C, 135, Y, and / or B. The meningococcal protein antigens can be selected from adhesins, auto-transporters, toxins, iron acquisition proteins, and membrane-associated proteins (preferably, integral proteins of the outer membrane). See also references 63-71.

Streptococcus pneumoniae: S. pneumoniae antigens include a saccharide (including a polysaccharide or an oligosaccharide) and / or the protein from S. pneumoniae. Protein antigens can be selected, for example, from a protein identified in any of references 72-77. The proteins of S. pneumoniae can be selected from the triad family of poly-histidine. (PhtX), the Choline Linker Protein family (CbpX), truncated CbpX, the LytX family, the truncated LytX, the truncated chimeric proteins of CbpX-truncated LytX, pneumolysin (Ply), PspA, PsaA, Spl28, SplOl, Spl30, Spl25 or Spl33. See also references 78-84.

Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcus antigens may include a protein identified in reference 85 or 86 (including GAS40), fragments fusions of GAS M proteins (including those described in references 87-89) ), fibronectin binding protein (Sfbl), proteins associated with the hemo-streptococcal group (Shp), and streptolysin S (SagA). See also references 85, 90 and 91.

Moraxella catarrhalis: Moraxela antigens include antigens identified in references 92 and 93, outer membrane protein antigens (HMW-OMP), C antigen, and / or LPS. See also references 94.

Bordetella pertussis: Pertussis antigens include pertussis holotoxin (PT) and filamentous hemaglutine (FHA) of B. pertussis, optionally also in combination with pertactin and / or the antigen of agglutinogens 2 and 3. See also references 95 and 96.

Staphylococcus aureus: S. aureus antigens include type 5 and 8 capsular polysaccharides of S. aureus optionally conjugated to non-toxic, recombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAXMR, or antigens derived from surface proteins, invasins (leucocidin) , kinases, hyaluronidase), surface factors that inhibit the engulfment phagocytic (capsule, protein A), carotenoids, catalase production, protein A, coagulase, clotting factor and / or toxins that damage the membrane (optionally detoxified) that lyses the membranes of eukaryotic cells (hemolysins, leukotoxin, leucocidin). See also reference 97.

Staphylococcus epidermis: The antigens of S. epidermidis include the slime-associated antigen (SAA).

Clostridium tetani (Tetanus): Tetanus antigens include tetanus toxoid (TT), preferably used as a carrier protein as a whole / conjugated with the compositions of the present invention.

Corynebacterium diphtheriae (Diphtheria): Diphtheria antigens include diphtheria toxin or detoxified mutants thereof, such as CRM197. Additionally, antigens capable of modulating, inhibiting or associating with the ribosylation of ADP are contemplated for the combination / co-administration / conjugation with the compositions of the present invention. These antigens of diphtheria can be used as carrier proteins.

Haemophilus influenzae: The antigens of H. influenzae include a type B saccharide antigen, or protein D [98].

Pseudomonas aeruginosa: Pseudomonas antigens they include endotoxin A, Wzz protein and / or outer membrane proteins, including F proteins of the outer membrane (OprF) [99].

Legionella pneumophila. Bacterial antigens can be derived from Legionella pneumophila.

Streptococcus agalactiae (group B streptococci): Group B streptococcal antigens include protein antigens identified in references 85 and 100-103. For example, the antigens include the GBS80, GBS104, GBS276 and GBS322 proteins.

Neisseria gonorrhoeae: Gonococcal antigens include Por (or porin) protein such as PorB [104], a transfer binding protein, such as TbpA and TbpB [105], an opacity protein (such as Opa), a modifiable protein by. reduction (Rmp), and preparations of external membrane vesicle (OMV) [106]. See also references 52-54 and 107.

Chlamydia trachomatis: C. trachomatis antigens include antigens derived from serotypes A, B, Ba and C (trachoma agents, a cause of blindness), serotypes Li, L2 and L3 (associated with lymphogranuloma venereum) and serotypes D-K C. trachomatis antigens may also include an antigen identified in references 103 and 108-110, including PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK (CT396), CT398, OmpH-like (CT242) , L7 / L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761). See also reference 111.

Treponema pallidum (syphilis): The antigens of syphilis include the TmpA antigen.

Haemophilus ducreyi (which causes chancroid): The Ducreyi antigens include the outer membrane protein (DsrA).

Enterococcus faecalis or Enterococcus faecium: Antigens include a trisaccharide repeat or other enterococcus-derived antigens provided in reference 112.

Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX, HopY and / or urease antigen. [113-123].

Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutinin of S. saprophyticus antigen.

Yersinia enterocolitica: Antigens include LPS [124] Escherichia coli: E. coli antigens can be derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhered E. coli (DAEC), enteropathogenic E. coli (EPEC), and / or enterohaemorrhagic E. coli strains (EHEC).

Bacillus anthracis (anthrax): Antigens of B. anthracis are optionally detoxified and can be selected from components A (lethal factor (LF) and edema factor (EF)), both of which can share a common B component known as the protective antigen (PA). See also references 125-127.

Yersinia pestis (plague): The antigens of the plague include the capsular antigen Fl [128], LPS [129], the antigen V [130].

Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins, LPS, BCG antigens, an 85B antigen fusion protein (Ag85B) and / or ESAT-β optionally formulated in cationic lipid vesicles [131], antigens associated with isocitrate-dehydrogenase of Mycobacterium tuberculosis (Mtb) [132], and / or PT51 antigens [133].

Rickettsia: Antigens include other membrane proteins, including protein A and / or B from the outer membrane (OmpB) [134], LPS, and surface protein antigen (SPA) [135].

Listeria monocytogenes: Bacterial antigens can be derived from Listeria monocytogenes.

Chlamydia pneumoniae: Antigens include those identified in references 108 and 136 to 141.

Vibrio cholerae: Antigens include proteinase antigens, particularly lipopolysaccharides of Vibrio cholerae II, specific polysaccharides of 01 Inaba 0, 0139 of V. cholerae, vaccine antigens of IEM108 [142], and / or Zonula occludens toxin (Zot ).

Salmonella typhi (typhoid fever): Antigens include capsular polysaccharides, preferably conjugates (Vi, for example, vax-TyVi).

Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (such as OspA, OspB, Osp C and Osp D), other surface proteins such as OspE-related proteins (Erps), decorin binding proteins (such as DbpA), and antagonistically variable VI proteins, such as the antigens associated with P39 and P13 (an integral membrane protein, [143]) and the antigenic variation protein VIsE [144].

Porphyromonas gingivalis: Antigens include the outer membrane protein (OMP). See also reference 145.

Klebsiella: Antigens include an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid.

Additional bacterial antigens may be capsular antigens, saccharide antigens or protein antigens of any of the foregoing. Additional bacterial antigens may also include an outer membrane vesicle preparation (OMV). In addition, the antigens include live, attenuated and / or purified versions of any of the aforementioned bacteria. The antigens used in the present invention may be derived from gram negative and / or gram positive bacteria. The antigens used in the present invention can be derived from aerobic and / or anaerobic bacteria.

B. Viral antigens Viral antigens suitable for use in the invention include inactivated (or killed) viruses, attenuated viruses, divided virus formulations, purified subunit formulations, viral proteins that can be isolated, purified or derived from a virus, and particles similar to viruses (VLPs). Viral antigens can be derived from viruses propagated on cell culture or other substrates. Alternatively, viral antigens can be expressed recombinantly. Viral antigens preferably include epitopes that are exposed on the surface of the virus during at least one stage of their life cycle. Viral antigens are preferably conserved through multiple or isolated serotypes. Viral antigens include antigens derived from one or more of the viruses described below, as well as the examples of specific antigens identified below.

Orthomyxovirus: Viral antigens can be derived from an Orthomyxovirus, such as Influenza A, B and C. Orthomyxovirus antigens can be selected from one or more of the viral proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein ( NP), matrix protein (MI), membrane protein (M2), one or more of the transcriptase components (PB1, PB2 and PA). The preferred antigens include HA and NA. Influenza antigens can be derived from interpandemic (annual) influenza strains. Alternatively, influenza antigens can be derived from strains with the potential to cause pandemic outbreaks (for example, strains of influenza with new haemagglutinin, compared to the inina haemagglut strains currently in circulation, or strains of the influenza that are pathogenic in avian subjects and have the potential to be transmitted horizontally in the human population, or strains of influenza that are pathogenic to humans).

Paramyxoviridae virus: Viral antigens can be derived from Paramyxoviridae virus, such as Pneumovirus (RSV), Paramyxovirus (PIV) and Morbilivirus (Measles). [146-148].

Pneumoviruses: Viral antigens can be derived from a Pneumovirus, such as respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkey rhinotracheitis virus. Preferably, the Pneumovirus is RSV. Pneumovirus antigens can be selected from one or more of the following proteins, including the fusion of surface proteins (F), glycoprotein (G) and protein small hydrophobic (SH), matrix and M2 proteins, nucleocapsid proteins N, P and L and non-structural proteins NS1 and NS2. Preferred Pneumovirus antigens include F, G and M. See for example, reference 149. Pneumovirus antigens can also be formulated in or derived from chimeric viruses. For example, the RSV / PIV chimeric viruses can comprise components of RSV and PIV.

Paramyxovírus: Viral antigens can be derived from a Paramyxovírus, such as Parainfluenza virus types 1 - 4 (PIV), mumps, Sendai virus, simian virus 5, bovine parainfluenza virus and Newcastle disease virus. Preferably, the Paramyxovírus is PIV or mumps. Paramyxovirus antigens can be selected from one or more of the following proteins: hemagglutinin-neuraminidase (HN), fusion proteins Fl and F2, nucleoprotein (NP), phosphoprotein (P), large protein (L), and matrix protein (M) Preferred Paramyxovirus proteins include HN, Fl and F2. Paramyxovirus antigens can also be formulated in or derived from chimeric viruses. For example, chimeric RSV / PIV viruses can comprise components of RSV and PIV. Commercially available vaccines for mumps include live attenuated mumps virus, either in a form monovalent or in combination with vaccines for measles and rubella (MMR).

Morbillivirus: Viral antigens can be derived from a Morbillivirus, such as measles. Morbillivirus antigens can be selected from one or more of the following proteins: hemagglutinin (H), glycoprotein (G), fusion factor (F), large protein (L), nucleoprotein (NP), phosphoprotein polymerase (P) , and matrix (M). Commercially available vaccines for measles include live attenuated measles virus, typically in combination with mumps and rubella virus (MMR).

Picornaviruses: Viral antigens can be derived from Picornaviruses, such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses and aftoviruses. Enterovirus-derived antigens, such as polioviruses, are preferred. See references 150 and 151.

Enteroviruses: Viral antigens can be derived from an enterovirus, such as poliovirus types 1, 2 or 3, Coxsackie virus A types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) types 1 to 9, 11 to 27 and 29 to 34 and enterovirus 68 to 71. Preferably, the enterovirus is poliovirus. The enterovirus antigens are preferably selected from one or more of the following capsid proteins VP1, VP2, VP3 and VP. Commercially available polio vaccines include the inactivated polio vaccine (IPV) and oral poliovirus vaccine (OPV).

Heparnavirus: Viral antigens can be derived from a Heparnavirus, such as the hepatitis A virus (HAV). Commercially available HAV vaccines include the inactivated HAV vaccine. [152,153].

Togavirus: Viral antigens can be derived from a Togavirus, such as a rubivirus, an alphavirus, or an arterivirus. Antibodies derived from rubivirus, such as rubella virus, are preferred. The togavirus antigens can be selected from El, E2, E3, C, NSP-I, NSPO-2, NSP-3 or NSP-4. The Togavirus antigens are preferably selected from El, E2 or E3. Commercially available rubella vaccines include a cold-adapted live virus, typically in combination with mumps and measles (MMR) vaccines.

Flavivirus: Viral antigens can be derived from a Flavivirus, such as tick borne encephalitis (TBE), dengue (types 1, 2, 3 or 4), fever yellow, Japanese encephalitis, West Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis, Powassan encephalitis. Flavivirus antigens can be selected from Pr, M, C, E, NS-I, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferably selected from PrM, M and E. The commercially available TBE vaccine includes inactivated virus vaccines.

Pestivirus: Viral antigens can be derived from a Pestivirus, such as bovine viral diarrhea (BVDV), classical pig fever (CSFV) or border disease (BDV).

Hepadnaviruses: Viral antigens can be derived from a Hepadnavirus, such as hepatitis B virus. Hepadnavirus antigens can be selected from surface antigens (L, M and S), core antigens (HBc, HBe). Commercially available HBV vaccines include subunit vaccines comprising the S protein of the surface antigen [153,154].

Hepatitis C virus: Viral antigens can be derived from hepatitis C virus (HCV). HCV antigens can be selected from one or more of El, E2, E1 / E2, NS345 polyprotein, NS 345 core polyprotein, nuclei and / or peptides of structural regions [155,156].

Rabdoviruses: Viral antigens can be derived from a Rabdovirus, such as a lisavirus (rabies virus) and vesiculovirus (VSV). Antibodies to rbdoviruses can be selected from glycoprotein (G), nucleoprotein (N), large protein (L), non-structural protein (NS). The vaccine for commercially available rabies virus comprises dead viruses developed on human diploid cells or fetal rhesus lung cells [157,158].

Caliciviridae: Viral antigens can be derivatives of Calciviridae, such as Norwalk virus and Norwalk-like viruses, such as the Hawai virus and the snowy mountain virus.

Coronaviruses: Viral antigens can be derived from a Coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine gastroenteritis virus (TGEV). Coronavirus antigens can be selected from barb (S), envelope (E), matrix (M), nucleocapsid (N), and / or glycoprotein hemagglutinin-esterase (HE). Preferably, the coronavirus antigen is derived from a SARS virus. Viral SARS antigens are described in reference 159.

Retroviruses: Viral antigens can be derived from a retrovirus, such as an oncovirus, a lentivirus or a foam virus. The oncovirus antigens can be derived from HTLV-I, HTLV-2 or HTLV-5. The lentivirus antigens can be derived from HIV-I or HIV-2. Retrovirus antigens can be selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigens can be selected from gag (p24gag and p55gag), env (gpl60, gpl20 and gp41), pol, tat, nef, rev vpu, miniproteins, (preferably p55 gag and deletion gpl40v). HIV antigens can be derived from one or more of the following strains: HIVJHB, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV- Reovirus: Viral antigens can be derived from a Reovirus, such as an orthoreovirus, a rotavirus, an orbivirus, or a coltivirus. The Reovirus antigens can be selected from the structural proteins ??, A2, A3, μ ?, μ2, s ?, s2, or s3, or non-structural proteins oNS, pNS, or ois. Preferred Reovirus antigens can be derived from a rotavirus. Rotavirus antigens can be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 and VP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, and / or NSP5. Preferred rotavirus antigens include VP4 (or the cleaved product VP5 and VP8), and VP7.

Parvovirus: Viral antigens can be derived from a Parvovirus, such as Parvovirus B 19. The Parvovirus antigens can be selected from VP-I, VP-2, VP-3, NS-I and / or NS-2. Preferably, the Parvovirus antigen is the VP-2 capsid protein.

Delta Hepatitis Virus (HDV): Viral antigens can be derived from HDV, particularly the HDV d antigen (see for example, reference 160).

Hepatitis E virus (HEV): Viral antigens can be derived from HEV.

Hepatitis G virus (HGV): Viral antigens can be derived from HGV.

Human herpesvirus: Viral antigens can be derived from a human herpesvirus, such as herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpesvirus human 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8). Human Herpesvirus antigens can be selected from immediate early proteins (a), early proteins (ß), and late proteins (?). HSV antigens can be derived from the HSV-I or HSV-2 strains. HSV antigens can be selected from glycoproteins gB, gC, gD and gH, fusion protein (gB), or immune escape proteins (gC, gE, or gl). VZV antigens can be selected from core, nucleocapsid, tegument, or envelope proteins. A live attenuated VZV vaccine is commercially available. EBV antigens can be selected from early antigenic proteins (EA), viral capsid antigen (VCA), and membrane antigen (MA) glycoproteins. CMV antigens can be selected from capsid proteins, envelope glycoproteins (such as gB and gH), and tegument proteins.

Papovaviruses: Antigens can be derived from Papovaviruses, such as papillomaviruses and polyomaviruses. Papillomaviruses include HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 and 65. Preferably , HPV antigens are derived from serotypes 6, 11, 16 or 18. HPV antigens can be selected from the capsid proteins (Ll) and (L2), or the -E7, or fusions thereof. HPV antigens are preferentially formulated into virus-like particles (VLPs). Polyavirus viruses include BK virus and JK virus. Polyomavirus antigens can be selected from VP1, VP2 or VP3.

C. Fungal Antigens Fungal or fungal antigens can be derived from one or more of the fungi described below. The fungal antigens can be derived from dermatofitros, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanu, Trichophyton concentricu, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton egnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubru, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoids, var. ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme. Fungal pathogens include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, capitatus Blastoschizomyces, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides im itis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Saccharomyces cerevisiae Saccharomyces, Saccharomyces boulardii , Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea sp. , Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp., Mucor spp., Absidia spp., Mortierella spp., Cunninghamella spp., Saksenaea spp., Alternaria spp., Curvularia spp., Helminthosporium spp., Fusarium spp., Aspergillus spp. , Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp. Processes for producing fungal antigens are well known in the art [161]. In a preferred method, a solubilized fraction extracted and separated from an insoluble fraction obtainable from fungal cells from which the cell wall has been substantially eliminated and / or at least partially eliminated, characterized in that the process comprises the steps of: obtaining the cells live fungal; get the fungal cells of which the cell wall has been substantially eliminated or at least partially eliminated; the explosion of the fungal cells from which the cell wall has been substantially eliminated or at least partially eliminated; obtaining an insoluble fraction; and the extraction and separation of a solubilized fraction from the insoluble fraction.

D. STD Antigens The compositions of the invention may include one or more antigens derived from a sexually transmitted disease (STD). Such antigens can provide prophylaxis or therapy for STDs such as chlamydia, genital herpes, hepatitis (such as HCV), genital warts, gonorrhea, syphilis and / or chancroid [162]. The antigens can be derived from one or more viral or bacterial STDs. Viral STD antigens for use in the invention can be derived from, for example HIV, herpes simplex virus (HSV-1 and HSV-2), human papillomavirus (HPV) and hepatitis (HCV). Bacterial STD antigens for use in the invention may be derived from, for example, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Escherichia coli, and Streptococcus agalactiae. Examples of specific antigens derived from these pathogens are described above. E. Respiratory Antigens The compositions of the invention may include one or more antigens derived from a pathogen that cause the respiratory disease. For example, respiratory antigens may be derived from a respiratory virus such as Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV), Morbillivirus (measles), Togavirus (rubella), VZV, and Coronavirus (SARS). Respiratory antigens can be derived from a bacterium that causes respiratory disease, such as Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are described above.

F. Pediatric Vaccine Antigens The compositions of the invention may include one or more antigens suitable for use in pediatric subjects. Pediatric subjects are typically less than about 3 years of age, or less than 2 years of age, or less than about 1 year of age. Pediatric antigens can be administered multiple times in the course of 6 months, 1, 2 or 3 years. Pediatric antigens can be derived from a virus that can be targeted to pediatric populations and / or a virus from of which pediatric populations are susceptible to infection. Pediatric viral antigens include antigens derived from one or more of Ortho-xovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV and mumps), Morbillivirus (measles), Togavirus (rubella), Enterovirus (polio), HBV, Coronavirus (SARS) , and the varicella zoster virus (VZV), Epstein Barr virus (EBV). Pediatric bacterial antigens include antigens derived from one or more of Streptococcus pneumoniae, Neisseria meningitidis, Streptococcus pyogenes (group A streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani (tetanus), Corynebacterium diphtheriae (diphtheria), Haemophilus influenzae type B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (group B streptococcus Streptococcus), and Escherichia coli. Examples of specific antigens derived from these pathogens are described above.

G. Suitable Antigens for Use in Elderly or Immunocompromised Individuals Compositions of the invention may include one or more antigens suitable for use in elderly or immunocompromised individuals. Such individuals may need to be vaccinated more frequently, with more doses high or with adjuvanted formulation to improve their immune response towards directed antigens. Antigens that can be targeted for use in elderly or immunocompromised individuals include antigens derived from one or more of the following pathogens: Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococci), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (tetanus), Cornynebacterium diphtheriae (diphtheria), Haemophilus influenzae type B (Hib), Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus agalactiae (group B streptococci), Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV and mumps), Morbillivirus (measles), Togavirus (rubella), Enterovirus (polio), HBV, Coronavirus (SARS), varicella zoster virus (VZV), Epstein Barr virus (EBV), Cytomegalovirus (CMV). Examples of specific antigens derived from these pathogens are described above.

H. Antigens Suitable for Use in Vaccines for Adolescents Compositions of the invention may include one or more antigens suitable for use in adolescent subjects. Teens may be in need of a reinforcement of a pediatric antigen, previously administered. Pediatric antigens that may be suitable for use in adolescents are described above. In addition, adolescents can be programmed to receive antigens derived from an STD pathogen in order to ensure protective or therapeutic immunity before beginning sexual activity. STD antigens that may be suitable for use in adolescents are described above.

I. Tumor Antigens One embodiment of the invention involves a tumor antigen or the cancerous antigen. Tumor antigens can be, for example, tumor antigens containing peptide, such as a polypeptide tumor antigen or glycoprotein tumor antigens. A tumor antigen may also be, for example, a tumor antigen containing saccharide, such as a glycolipid-like tumor antigen or a ganglioside tumor antigen. The tumor antigen may also be, for example, a tumor antigen containing a polynucleotide that expresses a tumor antigen containing polypeptide, for example, an RNA vector construct or a DNA vector construct, such as plasmid DNA. The tumor antigens appropriate for practice of the present invention encompass a wide variety of molecules, such as (a) tumor antigens containing polypeptides, including polypeptides (which may be, for example, 8-20 amino acids in length, although lengths outside this range are also common) , lipopolypeptides and glycoproteins, (b) tumor antigens containing saccharide, including polysaccharides, mucins, gangliosides, glycolipids and glycoproteins, and (c) polynucleotides expressing antigenic polypeptides. Tumor antigens can be, for example, (a) full length molecules associated with cancer cells, (b) homologs and modified forms thereof, including molecules with deleted, aggregated and / or substituted portions and (c) fragments thereof. Tumor antigens can be provided in recombinant form. Tumor antigens include, for example, antigens restricted to class I, recognized by CD8 + lymphocytes or class II restricted antigens recognized by CD4 + lymphocytes. Numerous tumor antigens are known in the art, including: (a) testicular cancer antigens such as NY-ESO-I, SSX2, SCP1 as well as polypeptides of the RAGE, BAGE, GAGE and MAGE families, for example, GAGE-I, GAGE-2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, target tumors of melanoma, lung, head and neck, NSCLC, breast, gastrointestinal and bladder), (b) mutated antigens, eg, p53 (associated with various solid tumors, eg, colorectal, lung cancer, head and neck), p21 / Ras (associated with for example, melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated for example with melanoma), MUM1 (associated for example with melanoma), caspase-8 (associated with, for example, head and neck cancer), CIA 0205 (associated for example, with bladder cancer), HLA-A2-R1701, beta catenin (associated with, for example, melanoma), TCR (associated with, for example, lymphoma of non-Hodgkins T cells), BCR-abl (associated with, for example, chronic myelogenous leukemia), triosaphosphate-isomerase, KIA 0205, CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example galectin 4 ( associated with, for example with colorectal cancer), galectin 9 (associated with, for example, Hodgkin's disease), pr otheinase 3 (associated with, for example, chronic myelogenous leukemia), WT 1 (associated with, for example, various leukemias), carbonic anhydrase (associated with, for example, kidney cancer), aldolase A (associated with, for example, lung cancer), PRAME (associated with, for example, melanoma), HER-2 / neu (associated with, for example, breast, colon, lung, and ovarian cancer), alpha-fetoprotein (associated with, for example, hepatoma), KSA ( associated with, for example cancer colorectal), gastrin (associated with, for example, pancreatic or gastric cancer), catalytic telomerase protein, MUC-I (associated with, for example, breast and ovarian cancer), G-250 (associated with, for example, carcinoma of cells kidney), p53 (associated with, for example, breast and colon cancer), and carcinoembryonic antigen (associated with, for example, breast cancer, lung cancer, and gastrointestinal tract cancers such as colorectal cancer), (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-1 / Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein / TRP1 and tyrosinase-related protein / TRP2 (associated with, for example, melanoma), (e) antigens associated with the prostate, such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated, for example, with prostate cancer, (f) Immunoglobulin idiotypes (associated with, by e example myeloma and B-cell lymphomas, for example), and (g) other tumor antigens, such as polypeptide and saccharide-containing antigens including (i) glycoproteins such as sialyl Tn and sialyl Lex (associated for example with breast and colorectal cancer ) as well as various mucins, the glycoproteins can be coupled to a carrier protein (for example, MUC-I can be coupled to KLH); (ii) lipopolypeptides (e.g., MUC-I bound to a portion lipid); (iii) polysaccharides (e.g., synthetic hexasaccharide Globe H), which can be coupled to carrier proteins (e.g., to KLH), (iv) gangliosides such as GM2, GM12, GD2, GD3, associated for example, with melanoma cerebral, lung cancer), which can also be coupled to carrier proteins (for example, KLH). Additional tumor antigens that are known in the art include Pl5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, antigens of Epstein Barr virus, EBNA, antigens of the human papillomavirus (HPV), including E6 and El, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23Hl, TAG- 72- 4, CA 19-9, CA 72-4, CA 17.1, NuMa, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125 , CA 15-3 (CA 27.29 \ BCAA), CA 195, CA 242, CA-50, CAM43, CD68 \ KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50 , MG7-Ag, MOV18, NB / 70K, NY-CO-I, RCAS1, SDCCAG16, TA-90 (protein binding to Mac-2 \ protein associated with cyclophilin C), TAAL6, TAG_72_, TLP, TPS, and Similar. These as well as other cellular components are described for example in reference 163 and in the references cited therein. Antigens containing polynucleotides according to the present invention typically comprise polynucleotides that code for cancerous antigens polypeptides such as those listed above. Agents containing polynucleotide include DNA or RNA, such as vector constructs, such as plasmid vectors (e.g., pCMV), which are capable of expressing cancerous, polypeptide antigens, in vivo. Tumor antigens can be derived, for example, from mutated or altered cellular components. After the alteration, the cellular components no longer perform their regulatory functions, and therefore the cell may experience uncontrolled growth. Representative examples of altered cellular components include ras p53, Rb, altered protein encoded by the Wilms tumor gene, ubiquitin, mucin, protein encoded by the DCC, APC, and CC genes, as well as the receptors such as receptor-like structures such such as neu, the thyroid hormone receptor, the platelet-derived growth factor receptor (PDGF) receptor, insulin receptor, epidermal growth factor receptor (EGF), and the colony stimulating factor (CSF) receptor. These as well as other cellular components are described, for example, in reference 164 and in the references cited therein. In addition, bacterial and viral antigens can be used in conjunction with the compositions of the present invention for the treatment of cancer. In particular, carrier proteins such as CRM197, Tetanus toxoid, or the antigen of Salmonella typhimurium can be used in conjunction / conjugation with the compounds of the present invention for the treatment of cancer. Therapies in combination with the cancerous antigen will show increased efficacy and increased bioavailability compared to existing therapies. Additional information on cancer or tumor antigens can be found, for example, in reference 165 (for example, Tables 3 and 4), in reference 166 (for example, Table 1) and in references 167 to 189 Immunization can also be used against Alzheimer's disease, for example, using Abeta as an antigen [190].

J. Antigen Formulations In other aspects of the invention, methods are provided for producing microparticles having adsorbed antigens. The methods comprise: (a) the provision of an emulsion by dispersion of a mixture comprising (i) water, (ii) a detergent, (iii) an organic solvent, and (iv) a biodegradable polymer selected from the group consisting of poly (α-hydroxy acid), a polyhydrobutyric acid, a polycaprolactone, a polyorthoester, a polyanhydride and a polycyanoacrylate. The polymer is typically present in the mixture at a concentration of about 1% to about 30% relative to the organic solvent, while the detergent is typically present in the mixture at a weight to weight ratio of the detergent to the polymer of about 0.00001: 1 to about 0.1: 1 (more typically about 0.0001: 1) to about 0.1: 1, about 0.001: 1 to about 0.1: 1, or about 0.005: 1 to about 0.1: 1); (b) removal of the organic solvent from the emulsion; and (c) the adsorption of an antigen on the surface of the microparticles. In certain embodiments, the biodegradable polymer is present at a concentration of about 3% to about 10% relative to the organic solvent. The microparticles for use herein will be formed from materials that are sterilizable, non-toxic and biodegradable. Such materials include, without limitation, poly (α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride, PACA, and polycyanoacrylate. Preferably, the microparticles for use with the present invention are derived from a poly (α-hydroxy acid), in particular, from a poly (lactide) ("PLA") or a copolymer of D, L-lactide and glycolide or glycolic acid , such as a poly (D, L-lactide-co-glycolide) ("PLG" or "PLGA"), or a copolymer of D, L-lactide and caprolactone. The microparticles can be derived from any of the various polymeric initial materials having a variety of molecular weights and, in the case of copolymers such as PLG, a variety of proportions' lactide: glycolide, the selection of which will largely be a matter of choice, depending in part on the co-administered macromolecule. These parameters are fully discussed below. Additional formulation methods and antigens, especially tumor antigens, are provided in reference 191.

Medical methods and uses Once formulated, the compositions of the invention can be administered directly to the subject. The subjects that are going to be treated can be animals; in particular human subjects can be treated. The compositions can be formulated as vaccines that are particularly useful for vaccines to children and adolescents. These can be distributed by systemic and / or mucosal routes. Typically, the compositions are prepared as injectables, either as suspensions or liquid solutions; Suitable solid forms for the solution in, liquid vehicles before injection can also be prepared. The direct distribution of the compositions in general will be parenterally, (for example, by injection, either subcutaneously, intraperitoneally, intravenously or intramscularly or distributed to the interstitial space of a tissue). The compositions may also be administered to a lesion. Other modes of administration include oral and pulmonary administration, suppositories and transdermal or transcutaneous applications (eg, see reference 192), needles and hyporrocies. The treatment of the dose can be a simple dose scheme or a multiple dose scheme (for example, including booster doses). The vaccines of the invention are preferably sterile. These are preferably pyrogen-free. These are preferably buffered, for example, at a pH between 6 and 8, generally around pH 7. Where a vaccine comprises an aluminum hydroxide salt, it is preferred to use a histidine buffer [193]. The vaccines of the invention may comprise detergent, (eg, a Tween, such as Tween 80) at low levels (eg, <0.01%). The vaccines of the invention may comprise a sugar alcohol (e.g., mannitol) or trehalose, for example, about 15 mg / ml, particularly if these are to be lyophilized. The optimal doses of the individual antigens can be evaluated empirically. In general, however, the saccharide antigens of the invention will be administered to a dose of between 0.1 ml and 100 μg of each saccharide per dose, with a typical dose volume of 0.5 ml. The dose is typically between 5 and 20 μ? per saccharide per dose. These values are measured as saccharide. The vaccines according to the invention can be either prophylactic (for example, to prevent infection) or therapeutic (for example, to treat the disease after infection), but will typically be prophylactic. The invention provides a conjugate of the invention for use in medicine. The invention also provides a method for producing an immune response in a patient, which comprises administering to a patient a conjugate according to the invention. The immune response is preferably protective against meningococcal disease, pneumococcal disease or H. influenzae and may comprise a humoral immune response and / or a cellular immune response. The patient is preferably a child. The method can produce a reinforcing response in a patient who has already been prepared against meningococcus, pneumococcus or H. influenzae. The invention also provides the use of a conjugate of the invention in the manufacture of a medicament for producing an immune response in a patient, wherein the patient has been pre-treated with a saccharide antigen different from that comprised within the composition. conjugated to a carrier. The invention also provides the use of a conjugate in the manufacture of a medicament for producing an immune response in a patient, wherein the patient has been pre-treated with the same saccharide antigen as that comprised within the composition conjugated to a different carrier. . The medicament is preferably an immunogenic composition (e.g., a vaccine). The medicament is preferably for the prevention and / or treatment of a disease caused by a Neisseria (e.g., meningitis, septicaemia, gonorrhea, etc.), by H. influenzae (e.g., otitis media, bronchitis, pneumonia, cellulitis, pericarditis, meningitis etc.) or pneumococci (eg, meningitis, sepsis, pneumonia, etc.). The prevention and / or treatment of bacterial meningitis is thus preferred. Vaccines can be tested in standard animal models (for example, see reference 194). The invention further provides a kit comprising: a) a first conjugate of the invention and b) a second conjugate of the invention. Adjuvants The conjugates of the invention can be administered in conjunction with other agents immunoregulators. In particular, the composition will usually include an adjuvant. Adjuvants that can be used in the compositions of the invention include but are not limited to: A. Compositions Containing Minerals Compositions containing minerals suitable for use as adjuvants in the invention include mineral salts, such as aluminum salts and calcium salts. Such mineral compositions may include mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, etc. [see for example, chapters 8 and 9 of reference 195], or mixtures of different mineral compounds (for example, a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of phosphate), with the compounds that it takes any suitable form (eg, crystalline gel, amorphous, etc.), and with adsorption to the one or more preferred salts. Mineral-containing compositions can also be formulated as a metallic salt particle [196]. The aluminum salts can be included in the compositions of the invention such that the dose of Al3 + is between 0.2 and 1.0 mg per dose. A typical aluminum phosphate adjuvant is the Amorphous aluminum hydroxyphosphate with a molar ratio of PO4 / AI between 0.84 and 0.92, included at 0.6 mg Al3 + / ml. The adsorption with a low dose of aluminum phosphate can be used for example 50 and 100 μg Al3 + per conjugate per dose. Where an aluminum phosphate is used and it is desired not to adsorb an antigen to the adjuvant, this is favored by the inclusion of the free phosphate ions in solution (for example, by the use of a phosphate buffer).

B. Oil Emulsions Oil emulsion compositions suitable for use as adjuvants with conjugates of the invention include squalene / water emulsions such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer) [Chapter 10 of reference 195; see also references 197-199]. MF59 is used as the adjuvant in the trivalent subunit vaccine of influenza virus FLUADMR. The MF59 emulsion advantageously includes citrate ions, for example 10 mM sodium citrate buffer. Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions. The preferred oil-in-water submicron emulsions for use herein are squalene / water emulsions optionally containing various amounts of MTP-PE, such as an oil-in-water submicron emulsion containing 4-5% w / v of squalene, 0.25-1.0% w / v of Tween 80 (polyoxyethylene sorbitan monooleate), and / or 0.25-1.0% of Span 85 (sorbitan triolate), and optionally, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanin-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE). Oil-in-water submicron emulsions, methods of making them and immunomodulating agents such as muramyl peptides, for use in the compositions, are described in detail in references 197 and 200-201. An emulsion of squalene, a tocopherol, and Tween 80 may be used. The emulsion may include buffered saline. This may also include Span 85 (for example 1%) and / or lecithin. These emulsions can have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene: tocopherol is preferably <; 1 since it provides a more stable emulsion. An emulsion of this type can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml of squalene), then microfluidizing the mixture. The resulting emulsion may have submicron oil droplets, for example, with an average diameter of between 100 and 250 nm, preferably of approximately 180 nm. An emulsion of squalene, a tocopherol and a Triton detergent (eg, Triton X-100) can be used. An emulsion of squalene, polysorbate 80 and poloxamer 401 ("Pluronic ^ L121") can be used. The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful distribution vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the adjuvant of "SAF-I" [202] (0.05-1% Thr-DP, 5% squalene, 2.5% of Pluronic L121 and 0.2% polysorbate 80). This can also be used without the Thr-MDP, as in the adjuvant "AF" [203] (5% squalene, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidization is preferred. Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants.

C. Saponin Formulations [Chapter 22 of Reference 195] The saponin formulations can also be used as adjuvants of conjugates of the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in bark, leaves, stems, roots, and even flowers of a wide range of plant species. Saponins isolated from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaparilla), Gypsophilla paniculata (bridal veil), and Saponaria officianalis (soap root). Formulations of saponin adjuvants include purified formulations such as QS21, as well as lipid formulations such as ISCOMs. QS21 is marketed as Stimulon ™. The saponin compositions have been purified using HPLC and RP-HPLC. The specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A production method of QS21 is described in reference 204. Saponin formulations may also comprise a sterol, such as cholesterol [205]. The combinations of saponins and cholesterols can be used to form unique particles called immunostimulation complexes (ISCOMs) [chapter 23 of reference 195]. ISCOMs also typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin in ISCOMs can also be used. Preferably, the ISCOM includes one or more of QuilA, QHA and QHC. The ISCOMs are also described in references 205-207. Optionally, ISCOMS can be provided with additional detergents [208]. A review of the development of saponin-based adjuvants can be found in references 209 and 210.

D. Virosomes and virus-like particles Virosomes and virus-like particles (VLPs) can also be used in adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. These are generally non-pathogenic, non-replicating and in general do not contain any native viral genome. Viral proteins can be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), hepatitis B virus (such as core or capsid proteins), hepatitis E virus, measles virus, Sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phages, phage < 2ß (such as coat proteins), GA-phage, fr-phage, phage AP205 and Ty (such as the pl protein of the retrotransposon Ty). The VLPs are discussed further in references 211-216. Virosomes are further discussed in, for example, reference 217.

E. Bacterial or Microbial Derivatives Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides and ADP ribosylating toxins and detoxified derivatives of the same. Non-toxic derivatives of LPS include monophosphoryl lipid A (PL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 des-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred form of "small particle" of de-O-acylated monophosphoryl lipid A is described in reference 218. Such "small particles" of 3dMPL are small enough to be sterilized by filtration through a 0.2 μm membrane. [218] Other non-toxic derivatives of LPS include monophosphoryl lipid A mimetics, such as the aminoalkyl glucosaminide derivatives for example RC-529 [219, 220]. Derivatives of lipid A include lipid A derivatives of Escherichia coli such as OM-174. OM-174 is described, for example, in references 221 and 222.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences that contain a CpG portion (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded ARs and oligonucleotides containing palindromic or poly (dG) sequences have also been shown as immunostimulators. The CpG's may include nucleotide / analog modifications such as phosphorothioate modifications and may be double-stranded or single-stranded. References 223, 224 and 225 describe possible substitutions of analogues, for example, the replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of the CpG oligonucleotides is further discussed in references 226-231. The CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT portion [232]. The CpG sequence may be specific to induce a Thl immune response, such as a CpG-A ODN, or it may be more specific to induce a B cell response, such as CpG-B ODN. The ODN of CpG-A and CpG-B are discussed in references 233-235. Preferably, the CpG is an ODN of CpG-A. Preferably, the CpG oligonucleotide is constructed so that the 5 'end is accessible for receptor recognition. Optionally, two oligonucleotide sequences of CpG can be linked in their 3 'ends to form "immunomers". See, for example, references 232 and 236-238. The bacterial ADP ribosylating toxins and the detoxified derivatives thereof can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (heat-labile enterotoxin "LT" of E. coli), cholera ("CT"), or pertussis ("PT"). The use of the detoxified ribosylating toxins of ADP as mucosal adjuvants is described in reference 239 and as parenteral adjuvants in reference 240. The toxin or toxoid is preferably in the form of a holotoxin, which comprises subunits A and B. Preferably , subunit A contains a detoxification mutation; preferably subunit B is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72 and LT-G192. The use of the ADP ribosylating toxins and the detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in references 241-248. The reference number for amino acid substitutions is preferably based on the alignments of subunits A and B of the ADP ribosylating toxins described in reference 249, specifically incorporated by reference herein in their entirety. The compounds of the formula I, II or III, or the salts thereof, can also be used as adjuvants: I? m as defined in reference 250, such as 'ER 803058', 'ER 803732', 'ER 804053', ER 804058 ',' ER 804059 ',' ER 804442 ',' ER 804680 ',' ER 804764 ',' ER 803022"or 'ER 804057' for example: F. Human immunomodulators Suitable human immunomodulators for use as adjuvants in the invention include cytokines, such as interleukins (e.g., IL-1, IL-2-, IL-4, IL-5, IL-6, IL-7, IL -12 [251], IL-23, IL-27 [252], etc.) [253], interferons (for example interferon y), macrophage colony stimulating factor, tumor necrosis factor, and macrophage inflammatory protein-lalpha (??? - lalfa) and MIP-lbeta [254].

G. Bioadhesives and Mucoadhesives The bioadhesives and mucoadhesives can also be used as adjuvants in the invention. Suitable bioadhesives include the esterified hyaluronic acid microspheres [255] or the mucoadhesives such as the crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof can also be used as adjuvants in the invention [256]. H. Microparticles Microparticles can also be used as adjuvants in the invention. The microparticles (e.g., a particle from -100 nm to -150 pm in diameter, more preferably -200 nm to -30 μp in diameter, and most preferably -500 nm to -10 μ in diameter) formed to from materials that are biodegradable and non-toxic (eg, a poly (α-hydroxy acid), a polyhydroxybutyl acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly (lactide-co-glycolide) are preferred , optionally treated to have a negatively charged surface (for example with SDS) or a positively charged surface (for example with a cationic detergent such as C ).

I. Liposomes (Chapters 13 and 14 of reference 195) Examples of liposomal formulations suitable for use as adjuvants are described in references 257-259.

J. Polyoxyethylene ether and polyoxyethylene ester formulations Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [260]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [261] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [262]. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl- ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP) PCPP formulations (poly [di (carboxylatophenoxy) fofazene]) are described, for example, in references 263 and 264.

L. Muramyl peptides Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanyl- D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine MTP-PE) .

M. Imidazoquinolone Compounds Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include Imiquamod and its homologs (eg "Resiquimod 3M"), further described in references 265 and 266.

N. Thiosemicarbazone Compounds The examples of thiosemicarbazone compounds, as well as the methods of formulation, manufacture and selection for the compounds, all suitable for use as adjuvants in the invention, include those described in reference 267. Thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as O. Tryptantrin Compounds Examples of triptantrin compounds, as well as methods for the formulation, manufacture and selection of compounds, all suitable for use as adjuvants in the invention, include those described in reference 268. The triptantrin compounds they are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-OI. P. Nucleoside analogs Various nucleoside analogs can be used as adjuvants, such as (a) Isatorabine (ANA- and the prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds described in references 269 to 271; (f) a compound having the formula: where: Ri and R2 are each independently H, halo, -NRa bf -OH, alkoxy of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms substituted, heterocyclyl, substituted heterocyclyl, aryl of 6 to 10 carbon atoms, aryl of 6 to 10 carbon atoms substituted, alkyl of 1 to 6 carbon atoms or alkyl of 1 to 6 carbon atoms substituted. R3 is absent, hydrogen, alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms substituted, aryl of 6 to 10 carbon atoms, substituted aryl of 6 to 10 carbon atoms, heterocyclyl or substituted heterocyclyl; R4 and R5 are each independently hydrogen, halo, heterocyclyl, substituted heterocyclyl, -C (0) -Rd, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms or linked together to form a ring of 5 members as in R4-5: being the union achieved in the links indicated by a ????? Xi and X2 are each independently N, C, 0, or S; R8 is hydrogen, halo, -OH, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, -OH, -NRaRb, - (CH2) n-0- Rc, -0- (alkyl of 1 to 6 carbon atoms), -S (0) pRe, or -C (0) -Rd; R9 is hydrogen, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms, heterocyclyl, substituted heterocyclyl or Rga, where Rga is: the junction being achieved at the bond indicated by a * ???? Rio and Rn are each independently hydrogen, halo, alkoxy of 1 to 6 carbon atoms, substituted alkoxy of 1 to 6 carbon atoms, -NRaRb, or - OH; each of Ra and Rb is independently hydrogen, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms, -C (0) Rd, aryl of 6 to 10 carbon atoms; each Rc is independently hydrogen, phosphate, diphosphate, triphosphate, alkyl of 1 to 6 carbon atoms or substituted alkyl of 1 to 6 carbon atoms; each Rd is independently hydrogen, halo, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, substituted alkoxy of 1 to 6 carbon atoms, -NH2, - NH (alkyl of 1 to 6 carbon atoms), -NH (substituted alkyl of 1 to 6 carbon atoms), -N (alkyl of 1 to 6 carbon atoms) 2, - (substituted alkyl of 1 to 6 carbon atoms) carbon) 2, aryl of 6 to 10 carbon atoms, or heterocyclyl; each Re is independently hydrogen, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms, aryl of 6 to 10 carbon atoms, substituted aryl of 6 to 10 carbon atoms, heterocyclyl, or substituted heterocyclyl; each Rf is independently hydrogen, alkyl of 1 to 6 carbon atoms, substituted alkyl of 1 to 6 carbon atoms, -C (O) Rd, phosphate, diphosphate or triphosphate; each n is independently 0, 1, 2 or 3; each p is independently 0, 1 or 2; or o (g) a pharmaceutically acceptable salt of any of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer.

Q. Lipids linked to the acyclic main chain containing phosphate Adjuvants containing lipids linked to an acyclic main chain containing phosphate, include the TLR4 antagonist, E5564 [272, 273]: R. Small molecule immunopotentiators (SMIPs) SMIPs include: • 2-methy1-1- (2-methylpropyl) -lH-imidazo [4, 5-c] quinolin-2,4-diamine; N2, N2-dimethyl-1- (2-methylpropyl) -lH-imidazo [4, 5 c] quinolin-2, -diamine; N2-ethyl-N2-methyl-1- (2-methylpropyl) -lH-imidazo [4,5- c] quinolin-2,4-diamine; • N2-methyl-l- (2-methylpropyl) -N2-propyl-lH-imidazo [4,5- c] quinolin-2,4-diamine; 1- (2-methylpropyl) -N2-propyl-lH-imidazo [4, 5-c] quinolin-2,4-diamine; • N2-Butyl-1- (2-methylpropyl) -lH-imidazo [4,5-c] quinoline- 2,4-diamine; N2-butyl-N2-methyl-1- (2-methylpropyl) -lH-imidazo [4, 5-c] quinolin-2,4-diamine; N2-methyl-1- (2-methylpropyl) -N2-pentyl-1H-imidazo [4,5-c] quinolin-2,4-diamine; N2-methyl-l- (2-methylpropyl) -N2-prop-2-enyl-lH-imidazo [4, 5-c] quinolin-2,4-diamine; 1- (2-methylpropyl) -2- [(phenylmethyl) thio] -1H-imidazo [4,5-c] quinolin-4-amine; 1- (2-methylpropyl) -2- (propylthio) -1H-imidazo [4,5-c] quinolin-4-amine; 2- [[4-amino-l- (2-methylpropyl) -1H-imidazo [4, 5-c] quinolin 2-yl] (methyl) amino] ethanol; 2- [[4-amino-l- (2-methylpropyl) -1H-imidazo [4, 5-c] quinolin-2, -yl] (methyl) amino] ethyl acetate; 4-amino-1- (2-methylpropyl) -1,3-dihydro-2H-imidazo [4,5-c] quinolin-2-one; N2-butyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [, 5-c] quinolin-2,4-diamine; N2-butyl-N2-methyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4, 5-c] quinolin-2,4-diamine; N2-methyl-1- (2-methylpropyl) -N4, 4-bis (phenylmethyl) -1H-imidazo [4, 5-c] quinolin-2, -diamine; N2, N2-dimethyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4, 5-c] quinolin-2,4-diamine; 1- . { -amino-2- [methyl (propyl) amino] -IH-imidazo [4,5- c] quinolin-1-yl} -2-methylpropan-2-ol; 1- [4-amino-2- (propylamino) -lH-imidazo [4, 5-c] quinolin-1-yl] -2-methylpropan-2-ol; · N4, N4-dibenzyl-l- (2-methoxy-2-methylpropyl) -N2-propyl-lH-imidazo [4, 5-c] quinolin-2,4-diamine.

S. Proteosomes An adjuvant is a proteosome preparation of the outer membrane, prepared from a first Gram-negative bacterium in combination with a preparation of liposaccharides derived from a second Gram-negative bacterium, wherein the preparations of the proteasome of the External membrane protein and liposaccharide form a non-covalent, stable adjuvant complex. Such complexes include "IVX-908", a complex comprised of the outer membrane Neisseria meningitidis and lipopolysaccharides. These have been used as adjuvants for influenza vaccines [274].

T. Other adjuvants Other substances that act as immunostimulatory agents are described in references 195 and 275. Additional useful adjunctive substances include: 5'-methylnosine monophosphate ("MIMP") [276].

A polyhydroxylated pyrrolizidine compound [277], such as one having the formula: where R is selected from the group consisting of hydrogen, linear or branched acyl, substituted or unsubstituted, saturated or unsaturated, alkyl (for example cycloalkyl), alkenyl, alkynyl and aryl, or a pharmaceutically acceptable salt or derivative thereof. Examples include, but are not limited to: casuarin, casuarin-6-D-glucopyranose, 3-epi-casuarin, 7-epi-casuarin, 3,7-diepi-casuarin, etc. · A gamma-inulin [278] or derivative thereof, such as algamulin. • The compounds described in reference 279. The compounds described in reference 280, which include: acylpiperazine compounds, indoldione compounds, tetrahydroisoquinoline compounds (THIQ), benzocyclodione compounds, aminoazavinyl compounds, aminobenzimidazole quinolinone compounds (ABIQ ) [281, 282], hydraphthalamide compounds, benzophenone compounds, isoxazole compounds, sterol compounds, compounds of quinazilinone, pyrrolo compounds [283], anthraquinone compounds, quinoxaline compounds, triazine compounds, pyrazolopyrimidine compounds, and benzazole compounds [284]. · Loxoribine (7-allyl-8-oxoguanosine) [285]. A formulation of a cationic lipid and a co-lipid (usually neutral) such as aminopropyl-dimethyl-myristoyryloxy-propanaminium-difitanoylphosphatiethanolamine bromide ("Vaxfect inMR") or aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide -dioleylphosphatiethanolamine ("GAP-DLRIE: DOPE"). Formulations containing (±) -N- (3-aminopropyl) -, -dimethyl-2, 3-bis (syn-9-tetradecenyloxy) -1-propanaminium salts are preferred [286]. The invention may also comprise combinations of aspects of one or more adjuvants identified above. For example, the following combinations can be used as adjuvant compositions in the invention: (1) a saponin and an oil-in-water emulsion [287]; (2) a saponin (for example QS21) + a non-toxic LPS derivative (for example 3dMPL) [288]; (3) a saponin (for example QS21) + a non-toxic LPS derivative (for example 3dMPL) + a cholesterol; (4) a saponin (for example QS21) + 3dMPL + IL-12 (optionally + a sterol) [289]; (5) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions [290]; (6) SAF, which contains 10% squalene, 0. 4% Tween 80, 5% pluronic block polymer L121, and thr-MDP, either microfluidized in a submicron emulsion or vortexed to generate an emulsion of larger particle size. (7) The RibiMR adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more components of the bacterial cell wall of the group consisting of monophosphorylid A (MPL), dimycolate of trehalose (TDM), and the cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL); and (9) one or more mineral salts (such as an aluminum salt) + an immunostimulatory oligonucleotide (such as a nucleotide sequence that includes a CpG portion).

Definitions The term "comprising" encompasses "including" as well as "consisting" for example a composition "comprising" X may consist exclusively of X or may include something additional such as X + Y. The term "approximately" in relation to to a numerical value x means, x ± 10%. All numerical values herein may be considered to be qualified by "approximately" unless the context otherwise indicates.

The word "substantially" does not exclude "completely" for example a composition that is "substantially free" from Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the various possible combinations of saccharide carrier and antigen (A) two monovalent conjugates, (B) a monovalent conjugate demonstrating that each carrier protein can be linked to more than one saccharide antigen molecule and ( C) a multivalent conjugate where more than one antigenically distinct saccharide is bound to each molecule of carrier protein. Figure 2 shows the antibody responses Anti-MenC IgG in serum. Groups of six BALB mice (c were immunized three times with decreasing amounts of N19-MenC or CRM-Menc (2.5, 0.625, 0.156 and 0.039 g of enC / dose) and 0.5 mg of aluminum hydroxide. serum were collected before (pre) and after (post-1, -2 and -3) of each immunization and tested individually to quantify MenC-specific IgG antibody titers.Each point represents the average antibody titer (± 1 SD) of each group at each time point Figure 3 shows the antibody responses Anti-carrier IgG in simple serum samples in mice immunized as described above. Since the mice were immunized with equal amounts of MenC in each conjugate, the final amount of the carrier protein is slightly different in the groups receiving CRM-enC and those receiving N19-Menc, due to the slight difference in the proportions of sugar to protein in the two constructions. Serum samples were collected before (pre-) and after (post-1, -2 and -3) of each immunization and individually tested to quantify carrier-specific IgG antibodies. Each point represents the average antibody titer (± 1 SD) of each group at each time point. Figure 4 shows the bactericidal activity in serum samples from mice immunized three times with decreasing amounts of N19-MenC or CRM-MenC (2.5 pg), 0.625 g, 0.156 μ ?, 0.039 pg MenC / dose) and 0.5 mg of aluminum hydroxide. The bactericidal antibody titers from the combined serum samples collected before (pre-) and after (post-1, -2, and -3) of each immunization, are shown. The results were expressed as reciprocal values of the highest serum dilution that gives more than 50% bacterial death. Figure 5 shows the antibody responses in anti-MenA and anti-MenC serum. Groups of six mice BALB / c were immunized three times with decreasing amounts of N19-MenA and N19-MenC either alone or in combination, or conjugates based on CRM (0.625, 0.156 and 0.039 and g of MenA and / or MenC / dose) in the presence of 0.06 mg of aluminum phosphate. Serum samples were collected before (pre-) and after (post-1, -2 and -3) of each immunization and anti-MenA and MenC specific IgG antibody titers were measured. Each point represents the average antibody titer (± 1 SD) of each group at each time point. Figure 6 shows the effect on the serogroup-specific antibody responses of a dose escalation of the tetravalent combined N19 conjugate vaccines. Groups of six BALB / c mice were immunized with decreasing amounts of N19-MenACWY (solid lines) or CRM-MenACWY (dashed lines) (from 2 to 0.074 ig of each MenPS / dose) in the presence of 0.06 mg of phosphate of aluminum as an adjuvant. Immunizations were performed on day 0, 21 and 35 and anti-MenA, anti-MenC, anti-MenW and anti-MenY serum anti-MenG antibody titers were measured after each immunization (post-1, -2 and -3). Each point represents the average antibody titer (± 1SD) of each group at each time point. Figure 7 shows the bactericidal activity against group C and W-135 in simple serum obtained from mice after two (post-2) and three (post-3) immunizations with 0.074 iq of each PS per dose (N19-MenACWY or CRM-MenACWY). Titers are expressed as reciprocal values of the highest serum dilution that gives at least 50% bacterial death. Each column represents the average titles (± SD) of the group at each time point. Figure 8 shows the dynamics of the avidity profile of anti-MenC antibodies generated in mice after immunization with NI 9-MenACWY or CRM-MenACWY as detailed in Materials and Methods. The high avidity IgG titers were measured on the combined sera by a modified ELISA method. The results are expressed in avidity index (AI) corresponding to the percentage of antibodies bound after elution with 75 mM NH4SCN from each group after each immunization (post 1st, post 2nd, post 3rd). Figure 9 shows the antibody responses against the carriers and their progenitor proteins in combined serum obtained after the third immunization. Each dot represents the antibody title of each group after three immunizations as described above. Figure 10 shows the antibody responses in anti-MenA serum. Groups of six BALB / c mice were immunized three times with decreasing amounts of tetravalent formulations prepared by mixing conjugated MenA to either N19 or CRM with MenCWY conjugated to either CRM or N19 (N19-MenA + CRM-MenCWY and vice versa CRM-MenA + N19-MenCWY). The control groups received tetravalent formulations containing a carrier (N19-MenACWY or CRM-MenACWY). The mice received increasingly smaller amounts of tetravalent formulations (from 0.67 μg to 0.074 g of each MenPS / dose) in the presence of 0.06 mg of aluminum phosphate as an adjuvant. For simplicity, only the results obtained after the highest immunization dose (0.67 and g) and the lowest (0.074 and g) are reported. Figure 11 shows the bactericidal activity in serum of BALB / c mice immunized three times with decreasing amounts of the bi-carrier or mono-carrier formulations as described above. Bactericidal antibody titers from combined serum samples collected after the second (post-2) and third (post-3) immunization were measured. The results were expressed as the reciprocal values of the highest serum dilution that gives more than 50% bacterial death. Figure 12 shows the anti-capsular IgG antibody responses. Groups of BALB / c or C57BL / 6 mice were immunized twice with N19-MenACWY or CRM-MenACWY (0.67 or 0.22 and g / dose of each MenPS) as conjugates, in the presence of 0.06 mg of aluminum phosphate. Were the serum samples were collected before (pre-) and after (post-1 and -2) of each immunization and the specific IgA antibody titers of MenA, MenC, Men, MenY were measured. Each point represents the average antibody titer (± 1 SD) of each group at each time point. Figure 13 shows the IgG anti-capsular antibody responses. Mice BALB / C H-2 d, BALB / B H-2 b, B10.BR H-2 k, B10-D2N H-2 and B10.D1 H-2 d were immunized three times with Ni 9-MenACWY or CRM -MenACWY (0.67 g / dose of each PS) in the presence of 0.06 mg of aluminum phosphate. Serum samples were collected before and after (post-1, -2 and -3) of each immunization and the specific IgA antibody titers of MenA, MenC, MenW, MenY were measured. Each bar represents the average antibody titer and the symbols correspond to the simple mouse of each group at each time point. Figure 14 shows the bactericidal activity in serum of mice with different genetic backgrounds immunized three times with Ni 9-MenACWY or CRM-MenACWY (0.67 g of each MenPS / dose) and 0.06 mg of aluminum phosphate. Bactericidal antibody titers from the combined serum samples collected after the third immunization (post-3) are shown. The results were expressed as the reciprocal values of the highest serum dilution giving more than 50% bacterial death.

Figure 15 shows the proliferation responses of specific T cells of the N19 epitope. Spleen cells from mice immunized three times with N19-MenACWY (6 N19 / dose) were tested to proliferate in vitro in the presence of 0.9-30 μ? of three individual peptides (P2TT, P23TT, P30TT) and 0.312 to 10 g / ml of protein N19, free or conjugated to PSs as indicated in the graph. The results were expressed as the stimulation index (SI) = (experimental cpm / antecedent cpm not stimulated). * = N19 concentrations of 0.312 to 10 and g / ml. Figure 16 shows the T-cell proliferation responses specific to the N19 epitope. Spleen cells from mice immunized twice with N19- in ACWY (6 μg N19 / dose) were tested to proliferate in vitro in the presence of 0.12-30 μ? of individual peptides (P2TT, P21TT, P23TT, P30TT, P32TT, HA, HBsAg) and 0.004 to 1 μ of N19 as indicated in the graph. The results were expressed as the stimulation index (SI) = (experimental cpm / antecedent cpm not stimulated). * = N19 concentrations from 1 to 0.004 μ ?. Figure 17 shows the pr o 1 i f e r a t i va response of T cells from congenic strains of mice immunized with N19-MenAC Y. Strains of mice with different H-2 haplotype were immunized three times with N19-MenAC Y (6 μg of N19 / dose) in the presence of 0.06 mg of aluminum phosphate. The spleen cells were tested to proliferate in vitro in the presence of 1.7-15 μ of N19 peptides (listed in Table 1) and 0.1 to 10 g / ml of N19 protein, free or conjugated to the SPs. The results were expressed as the stimulation index (SI) = (experimental cpm / antecedent cpm not stimulated). A SI < 2 was considered pos i t i o. Figure 18 shows the activation of cells T specific for P23TT, HA and HBsAg. The stimulation indices for the homologous peptides and the N19 protein, as determined in the proliferation assay. Groups of three mice were immunized at the base of the tail with a volume of 50 μ? containing 50 pg of the individual peptide emulsified 1: 1 in CFA. Seven days later lymph nodes and LN cells tested for their ability to proliferate in the presence of the homologous peptide or the N19 protein at different concentrations were removed. The results were obtained in triplicate cultures of a simple mouse. The results were expressed as the stimulation index (SI) calculated from the average cpm of the group experienced in 1 / cpm of the antecedent.

DETAILED DESCRIPTION OF THE INVENTION 1. Preparation of glycoconjugate 1.1 Expression and purification of polyepitopic protein NI 9 The E. coli strains possessing the recombinant plasmids pQE-Nl9 were developed 0 / N on LB agar plates, 100 μq / ml of ampicillin at 37 ° C. The developed bacteria were then inoculated in 500 ml of LB medium, 100 μg / ml of ampicillin and developed O / N at 37 ° C. 500 ml were then diluted in 5 liters of medium in a thermenator. Growth has been conducted under optimized conditions. When an OD6oonm value of 4.2 was obtained, the expression of the polyepitopic protein was induced by 3.5 hours by the addition of 1 mM IPTG (iso-propyl-thio-galactoside) to an OD6oonm of 7.2. Two samples of the bacterial culture supernatant were collected, at time zero before adding IPTG (t0 OD .2) and the final time point of expression (OD 7.2 tend). The obtained button was resuspended in sample buffer and loaded on a 12.5% SDS-PAGE in serial dilution corresponding to different ODs of bacterial culture. The whole bacterial culture was centrifuged at 5000 g in a JA10 rotor (Beckman, Fullerton, CA) for 20 minutes at 4 ° C. The cell button obtained from 60 g was suspended in 500 ml of lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2P04, 2 mM TCEP (Pierce) pH 8, stirred for 1 hour at room temperature and then incubated for 1 hour at 37 ° C. The supernatant containing the dissolved protein was collected by centrifugation at 12000 rpm in a J20 rotor (Beckman) for 20 minutes at room temperature, and subjected to Immobilized Metal Affinity Chromatography (IMAC). Before the adsorption of the sample on the IMAC column, 1 mM TCEP (tris (2-carboxyethyl) phosphine hydrochloride, Pierce) had been added, which previously showed that it was essential during the purification, to avoid co-purification of the pollutants covalently linked to N19 by disulfide bonds. The dissolved material was loaded on an XK50 column containing 360 ml of IDA (iminodiacetic acid) activated with nickel, Chelating Sepharose Fast Flow (Pharmacia, Uppsala, Sweden), the column was then washed with 5 volumes of lysis buffer A gradient of 300 ml was then applied from 6 M guanidine hydrochloride pH 8 to the 8 M urea pH 8 containing 1 mM TCEP. The column was washed with three volumes of buffer B (8 M urea, 100 mM NaH2P04, pH 7) and the proteins were eluted with 1800 ml of 0-200 mM imidazole gradient in buffer B. The fractions collected from the column were qualitatively analyzed on 12.5% SDS-PAGE (BioRad) and quantitatively by the Bradford protein determination method (test of Bio-Rad protein). The selected gradient fractions containing the purified recombinant proteins were subjected to Cation Exchange Chromatography (CEC). The combined fractions of 600 ml were loaded onto an XK50 column containing 120 ml of SP-Sepharose Rapid Flow resin (Pharmacia, Uppsala, Sweden). The column was washed with 5 volumes of buffer C (urea 7, 20 mM NaH2P04 pH 7, 10 mM β-mercaptoethanol) and the proteins were eluted with 1300 ml of 0 to 500 mM sodium chloride gradient in C buffer. gradient fractions containing the purified recombinant proteins, selected by 12.5% SDS-PAGE analysis (BioRad) were combined and dialysed against 10 mM NaH2P04, 150 mM sodium chloride, 10% glycerol. The final protein concentration was determined by the micro BCA method according to the manufacturer's instructions (Pierce). The protein was analyzed on 12.5% SDS-PAGE (BioRad). The optical density of the bands has been measured for the integrity evaluation (Computer Program Image Master ID Elite v4.00 LabScan). The level of endotoxins in the final protein preparation was determined by the kinetic tubidimetric method of amoebocyte limulus lysate (LAL) by the Quality Control Department (Chiron Vaccines Siena). 1. 2 Production of oligosaccharides The meningococcal polysaccharides of group A, C, W, Y were purified from strains of Neisseria meningitidis by standard procedure described for the production of meningococcal vaccine (291). The purified capsular polysaccharides were then depolymerized and activated in order to be coupled to the carrier protein as previously described (292, 293). In summary, the method for the preparation of serogroup C meningococcal oligosaccharide is described herein. The purified MenC capsular polysaccharide was subjected to hydrolysis in 10 mM sodium acetate buffer 5.0 to reduce the average degree of polymerization (DP). The reaction is conducted at 80 ° C for -12 hours until a DP of 10 was reached. The DP was followed online during the hydrolysis by analysis of the total sialic acid content in the initial polysaccharide solution (constant during hydrolysis) and the formaldehyde released from the terminal group of each chain after oxidation. This real-time PD measurement allowed extrapolation of the final hydrolysis time. The oligosaccharides were sized by Q-Sepharose FF ion exchange chromatography which retained the highest molecular weight polysaccharides on the column, while the lower molecular weight oligosaccharides (DP <6) were eluted from the column with mM sodium acetate buffer, 100 mM sodium chloride, pH 6.5. The desired oligosaccharide fraction was then eluted with 0.7 M tetrabutylammonium bromide ( ), a positive counter ion, which displaced the negatively charged oligosaccharides from the column. The products were then subjected to concentration / diafiltration against water on a 3K cut-off membrane to remove the excess TAB and to concentrate the MenC oligosaccharide in the preparation. After diafiltration, the retentate was dried by a rotary evaporation step. After this, the MenC oligosaccharide was subjected to reductive tuning to produce an oligosaccharide with a terminal primary amino group. The reaction mixture was made up to 10% DMSO, 90% methanol, 50 mM ammonium acetate, and 10 mM sodium cyanoborohydride, and incubated for 22 hours in a water bath covered at 50 ° C. The reaction mixture was then subjected to a rotary evaporation step to reduce the methanol content of the tuning reaction mixture, to avoid possible interaction with the silicon tubing and the diafiltration membranes in the next diafiltration step. The aminated oligosaccharides were then purified from the reagents (cyanoborohydride, DMSO, methanol) by concentration / diafiltration against 8 volumes of 0.5 M sodium chloride, followed by 4 volumes of 20 mM sodium chloride. The purified aminated oligosaccharides were dried to empty in preparation for the activation step. The MenC oligosaccharide was solubilized in water followed by the addition to the DMSO mixture. Triethylamine (TEA) was added to ensure sufficient deprotonation of the primary amino group of the oligosaccharide and the di-N-hydroxysuccinimide ester (bis-NHS) of the adipic acid. The bis-NHS was added in molar excess to favor the formation of the covalent bond of a simple oligosaccharide polymer to each molecule of the bis-NHS ester. The activated oligosaccharide was precipitated by the addition of acetone to the reaction mixture, which was also used to separate the oligosaccharides from DMSO, the bis-NHS ester and the TEA. The precipitate was dried under vacuum, weighed and stored at -20 ° C until use for conjugation. The procedure for the purification of the others PSs were basically the same, with minor modifications in reaction time and temperature [294]. 1. 3 Conjugation of N19 to meningococcal oligosaccharides After purification, size sorting and activation, the oligosaccharides were used for subsequent conjugation to the N19 protein [295]. Prior to the start of the conjugation experiment, the potential specific adsorption of the polysaccharides to the nickel-activated resin was preliminarily evaluated. In a typical conjugation experiment, 343.2 nmol of the carrier protein N19 were dissolved in guanidine hydrochloride pH 8, Na2P0, and adsorbed to a 5 ml nickel-activated Sepharose Fast Flow resin, pre-packaged (Pharmacia, Uppsala, Sweden) balanced in the same shock absorber. Guanidine hydrochloride was removed by washing the resin with 50 ml of 100 mM phosphate buffer, pH 7.5, and then 1 ml of 100 mM phosphate buffer pH 7.5 containing 6864 nmol of the activated mengococcal oligosaccharide (MenA , MenC, MenW or MenY) to the column, recirculating at room temperature for 2 hours. The column was washed with 50 ml of 100 mM Na2P04 pH 7.5 to remove excess unconjugated oligosaccharide. Finally, the conjugate was eluted with 300 mM imidazole, pH 7, 100 mM Na2P04 and analyzed on 7.5% SDS-PAGE. The selected fractions containing the conjugate were combined and dialysed against PBS. The glyco-conjugates were analyzed for the sugar and protein content. The saccharide content of the MenC, MenW and MenY conjugates was quantified by the determination of sialic acid (143), whereas that of the MenA conjugate was determined by the chromatographic determination of mannosamine-1-phosphate (121). The protein content was measured by the micro BCA assay (Pierce, Rockford, IL). The degree of glycosylation was calculated from the proportion of sugar to protein in weight. The conjugate vaccines based on CRM (CRM-MenA, CRM-MenC, CRM-enW, CRM-enY) taken as reference in this study were prepared by the Manufacturing Department (Chiron Vaccines Siena). 2. Mouse strains Unless otherwise specified, groups of six BALB / c mice from 7-week-old females were used. In another experiment, four congenic strains of 7-week-old female mice with the following H-2 haplotype were used: BALB / B (H-2b) congenital with BALB / c (H-2d) and B10.BR (H- 2k), B10.D2N (H-2q), B10.D1 (H-2d) congenic with C57BL / 6 (H-2b). The mice were purchased from Charles River (Calco, Italy) or Jackson Laboratories (Bar Harbor, Maine). 3. Schemes and immunization formulations in mice The mice were immunized subcutaneously on days 0, 21 and 35 with the N19 or CRM conjugates, with different formulations of 0.5 ml of monovalent, bivalent, tetravalent or bi-carrier conjugate vaccine, based on the content of saccharides diluted in 0.9% sodium chloride buffer as specified below. The individual serum samples were taken on days -1 (pre), 20 (post-1), 34 (post-2) and 45 (post-3) and frozen at - ° C until use. Spleens were harvested from mice immunized with N19 conjugates to evaluate T cell proliferation as described in the cell-mediated immune response section. 3. 1 Monovalent meningococcal C conjugate vaccine Mice were immunized with decreasing amounts of N19-Menc or CRM-MenC (from 2.5 to 0.039 ytg MenC / dose) in the presence of 0.5 mg of aluminum hydroxide as an adjuvant. Antibody titers were measured as described below. The conjugate containing N19 was more immunogenic than that with CRM (Figure 2). After two immunizations the N19-based constructs induced anti-MenC IgG antibodies in serum, at titers significantly higher than those induced by three doses of the CRM-MenC conjugate (eg post-2 N19-MenC at 0.625 μg versus post -3 CRM-MenC at 0.625 ig [P <0.01], post-2 N19-MenC at 0.156 \ ig versus post-3 CRM-MenC at 0.156 iq [P <0.05]). In addition, after three doses, minor amounts of the N19 conjugate were sufficient to induce anti-MenC IgG antibodies, significantly higher than those induced by the CRM-MenC conjugate (for example N19-MenC at 0.156 μg versus CRM-MenC at 0.625). μg [P < 0.01]).

Two and three immunizations with CRM-based conjugates induced strong anti-carrier antibody responses against CRM, even at the lowest dose levels tested (eg, 0.3 g and lower). In contrast, the specific antibody response of N19 was always negligible and was detectable (albeit at very low titers) only at the highest dose (for example 6 and g) (Figure 3). These low-grade anti-N19 antibodies did not recognize tetanus toxoid in solid phase. These results clearly show that the strong cooperative effect of polyepitolope N19 is not accompanied by the induction of significant levels of antibodies for itself or for native proteins. Since the protective immunity against MenC relies mainly on bactericidal antibodies that kill bacteria in the presence of complement, the functional activity of the induced antibodies was measured. According to the results obtained in ELISA, Figure 4 shows that the N19 conjugates were able to induce bactericidal antibodies at lower immunization doses than those used with the CRM-based conjugates. It is well known that after an immunization at the highest dose, the N19-MenC conjugate induced bactericidal antibodies with titers similar to those induced by two doses of the conjugate CRM-MenC. Mice immunized twice with lower amounts of N19-MenC produced higher bactericidal antibody titers than those immunized with CRM-MenC. These mice immunized with CRM-MenC required a third dose to achieve bactericidal antibody titers, comparable to those induced by the N19 conjugates. Therefore, NI 9 showed that it behaves as a stronger carrier than CRM by inducing antibodies with substantial functional activity against MenC after fewer injections with less dose. 3.2 Conjugate Vaccine of Bivalent Meningococcal AC The mice were immunized with N19-MenA and Nl9-MenC separately, and combined or CRM-MenA and CRM-MenC separated and combined (0.625, 0.156 or 0.039 pg of each MenPS / dose) in the presence of 0.06 mg of aluminum phosphate as adjuvant. Antibody titers were measured by ELISA as described below. As shown in the upper panel in Figure 5, co-administration of the MenA and MenC conjugates containing either the N19 or CRM carrier, the immunogenicity against MenA was accompanied as expected, by a significant reduction compared to that of the conjugates given alone (for example to 0.156 pg of post-2 NI 9-MenA versus N 19 -MenAC [P < 0.05] to 0.625 of post-3 NI 9-MenA versus NI 9-MenAC [P < 0.05]; to 0.625 versus post-2 CRM-MenA versus CRM-MenAC [P < 0.05]; at 0.156 μ? versus post-3 CRM-MenA versus CRM-MenAC [P < 0.05]). However, both bivalent formulations containing the N19 or CRM carrier induced comparable antibody titres (not statistically different) against MenA after two and three immunizations. The N19 carrier in monovalent and bivalent conjugate vaccines was able to induce a faster antibody response against MenA, producing an antibody response already after the first dose, whereas the CRM conjugates did not induce any measurable antibody titre. Also after two injections of the N19 conjugates the tendency was to promote a higher antibody response than the CRM conjugates, but the differences became statistically significant only at the lowest given dose of the monovalent vaccine (for example at 0.039 g. post-2 N19-MenA versus CRM-MenA [P <0.05] When the anti-MenC antibody response was measured (lower panel in Figure 5), no decrease in titers was observed after two or three doses When the monovalent and divalent formulations were compared, the decrease in the immunization dose was abolished after administration. anti-MenC antibody levels of the monovalent vaccine, obtained with the CRM conjugates, while those obtained with the N19 conjugates remained stable. Comparison of the titres obtained after an immunization with the bivalent vaccines, the CRM conjugates showed that they are unable to produce a substantial anti-MenC antibody response, whereas the N19 conjugates induced higher levels with a dose- answer. 3. 3 ACWY conjugated vaccine of tetravalent meningococcus The tetravalent formulations were prepared by mixing together an equivalent quantity of saccharide NI 9-MenA, N19-MenC, N19-Men and N 19 -MenY (N19-MenACWY). As a reference, clinical grade batches of the CRM conjugate vaccine (Chiron Vaccines, Siena) formulated before use were used when mixing the liquid CRM-MenCWY to the lyophilized CRM-MenA. The mice received increasingly smaller amounts of tetravalent formulations (from 2 g to 0.074 μ of each MenPS / dose) in the presence of 0.06 mg of aluminum phosphate as an adjuvant. Figure 6 shows for any of the four capsular polysaccharides of the serogroup and all Given the doses given, two or three immunizations with N19-MenACWY produced similar IgG titres. When comparing the antibody responses to the CRM conjugates after three immunizations, and those of the N19 conjugates after only two immunizations, no significant differences were found for all four serogroups. After the second dose, antibody titers against serogroups A and C when conjugated to N19 were significantly higher compared to those obtained when conjugated to CRM (anti-MenA IgG and anti-MenC: post-2 all the given doses N19 versus CRM: P <0.05). The N19 conjugates induced the production of antibody against the four polysaccharides after the primary immunization, whereas the CRM conjugates did not. In particular against MenC, as shown in panel B of Figure 6, significantly higher antibody titers were obtained with the N19 conjugates at all given doses (post-1 at all given doses N19 versus CRM: [P < 0.05]). The titers against MenA and MenW shown in panel A and C, were significantly higher at the highest dose when the N19 conjugates were administered once (at 2 \ iq post-1 N19 versus CRM: [P <0.05]) . The antibodies induced by both conjugates were predominantly IgG1 (data not shown). Importantly, it was perceived that the number of responding mice was higher when they were immunized with the N19 conjugates than with the CRM conjugates, especially after the first and second doses, whereas after the third dose all the mice responded. (Table 2).

Table 2: Percentage of mice responding to the four PS antigens (MenACWY) of each group. % of mice responding to PS MenA MenC Men MenY Post 1 Dosage PS NI9 CRM N19 CRM N19 CRM N19 CRM 2 pg 83 0 100 0 83 0 100 33 0. 67 μg 67 17 100 0 67 17 100 17 0. 22 g 17 0 83 0 50 0 50 33 0. 074 μg 0 0 83 0 0 17 67 17 Post 2 Dosage PS N19 CRM N19 CRM N19 CRM NI 9 CRM 2 g 100 67 100 100 100 83 100 100 0. 67 μg 100 50 100 83 100 83 100 83 0. 22 μg 100 83 100 100 100 100 100 100 0. 074 μg 100 33 100 67 100 83 100 100 Post 1 Dosage PS N19 CRM N19 CRM NI9 CRM N19 CRM 2 g 100 83 100 100 100 83 100 100 0. 67 ug 100 83 100 83 100 67 100 83 0. 22 pg 100 100 100 100 100 100 100 100 0. 074 g 83 100 100 100 100 67 100 100 N19-MenACWY was highly effective in inducing bactericidal antibodies against the four Men polysaccharides. In particular, the bactericidal titers against group C were significantly higher at all doses given a two doses of the N19 conjugates than the CRM conjugates. To raise the dose, the potency of the N19 carrier was revealed, since by limiting the dose, the N19 conjugates induced higher titers of bactericidal antibody against the four polysaccharides than those induced by the CRM conjugates. Bactericidal titers against MenC and MenW on simple sera from mice immunized at the lowest dose (0.074 pg) were analyzed in particular. Figure 7 shows that as for the ELISA titers, also the Bactericidal Serum Antibody (SBA) titers obtained with N19 conjugates were comparable a two or three doses. The bactericidal titers against MenC were already significantly higher a two immunizations with the N19 conjugates than those obtained a three injections of the CRM conjugates (anti-MenC SBA: post-2 N19 versus post-3 CRM: [P < 0.05]). The comparison of the bactericidal titers against MenW a two doses or a three obtained with either N19-based or CRM-based conjugates, found that N19 conjugates induced bactericidal antibody titers, significantly higher (SBA anti-MenW post-2 N19 versus CRM: [P < 0.05]; post-3 N19 versus CR: [P < 0.05]). A detailed analysis of the functional activity of Group A and C antibodies was conducted using a modified antigen binding assay that only measures high affinity antibodies [296]. The results show in Figure 8 that the antibodies obtained against MenC with 2 iq of N19 conjugates were already high avidity a a dose. Two immunizations were sufficient to induce efficient avidity maturation of almost all antibodies. The other groups, immunized with either lower amounts of the N19 conjugates or with the CRM conjugates, showed a similar maturation profile, with an increase from the baseline to approximately 50% of the high avidity antibodies only. a two doses (for simplicity only the groups immunized with the highest dose and the lowest dose are shown). To evaluate the influence of the carrier protein shared by four polysaccharides to induce the antibodies against itself, the antibodies against the carrier proteins used were measured (Figure 9). In addition, it was analyzed whether or not the antibodies raised against the carriers were capable of also binding to the progenitor proteins. Figure 9 shows in panel A that antibodies produced with CR conjugates likewise recognized either DT, the progenitor protein of CRM. In contrast, antibodies to the N19 conjugates did not cross-react with their parent proteins, such as tetanus toxoid (TT) and influenza hemagglutinin (HA), from which the N19 epitopes were derived. It should be noted that the ten epitopes (five repeated twice) of TT are contained in N19, representing more than 50% of its sequence. 3. 4 ACWY meningococcal tetravalent bi-carrier conjugate vaccine The tetravalent formulations were prepared by mixing conjugated MenA to either N19 or CRM with conjugated MenCWY either to CRM or N19 (N19-MenA + CRM-MenCWY and vice versa CRM-MenA + N19- MenC Y). The control groups received tetravalent formulations containing a carrier (N19-MenACWY or CRM-MenAC Y). The mice received increasingly smaller amounts of tetravalent formulations (from 0.67 μg to 0.074 μl of each of the Men / dose polysaccharides) in the presence of 0.06 mg of aluminum phosphate as an adjuvant. Antibody titers were determined using the methods described below. N19-MenAC Y produced, after the first dose, anti-MenA titers comparable to those obtained after Two doses of the vaccine based on CRM (Figure 6). In addition, mice immunized twice with the N19 conjugates promoted significantly greater bactericidal titers against MenA, than those immunized with the CRM conjugates (Figure 10). It was observed that when N19-MenA was administered simultaneously with CRM-MenCWY or vice versa, exchanging the carrier on MenA, the antibody response was significantly increased compared to the tetravalent formulation containing a single carrier (eg post-2 to 0.67 iq). : NI 9-Mena + CRM-MenCWY versus NI 9-MenACWY: [P <0.05]; post-3 to 0.67 g: N 19-MenA + CRM-MenCWY versus CRM-MenACWY: [P <0.01]; post -2 to 0.22 yg: CRM-MenA + N 19 -MenCWY versus CRM-MenACWY: [P <0.001]). However, it was noted that upon decreasing the immunization dose of both bi-carrier formulations, the anti-MenA antibodies decreased significantly in the IgG titer but not in their bactericidal cycle nor after two or after three immunizations (Figure 10). ). In addition, both bi-carrier vaccines induced comparable bactericidal titers at all doses (Figure 11). It is well known that the presence of N19 in all formulations consistently evoked an antibody response only after an immunization, whereas CRM alone did not (Figure 10). 3. 5 Mouse strains with different genetic backgrounds In a preliminary experiment, two groups of BALB / c and C57BL / 6 mice were immunized twice with 0.67 or 0.22 g of NI 9-MenACWY or CRM-MenACWY with 0.06 mg of aluminum phosphate. In another experiment, congenic strains of mice were immunized three times with tetravalent formulations NI 9-MenACWY or CRM-MenACWY (0.67 iq of each Men polysaccharide / dose) in the presence of 0.06 mg of phosphate prepared as described above. The BALB / c mice were used as control. Based on the previous results obtained in the BALB / c mice, it was decided to immunize mice only twice with two different doses of the tetravalent formulations containing N19 or CRM and the antibody responses against the four polysaccharides were measured (Figure 12). Again, it was evidenced in BALB / c mice that N19 behaved as a stronger carrier than CRM in the tetravalent vaccine, inducing in particular anti-MenA antibodies (BALB / c 0.22 pg post 2 N19 versus CRM: [P < 0.001 ]). It was observed that both conjugates containing N19 or CRM were less immunogenic in C57BL / 6 mice than in BALB / c mice, and the antibody responses were more variable. In addition, the best carrier effect of N19 was less evident against the four polysaccharides than that observed in BALB / c. However, the N19 conjugates were able to of consistently promoting antibody titers against the four polysaccharides already after the first immunization, while the CRM conjugates did not. As shown in Figure 13, the N19 and CRM conjugates were more immunogenic against the four conjugates in the BALES / c H-2d and B10.D1 H-2q strains. In general, more mice responded when they were immunized with N19-based conjugates that based on CRM. B10.D2N H-2d with the same haplotype as BALB / c mice, were better containers for the N19 conjugates than for the CRM conjugates. On the one hand, the BALB / B H-2b mice congenic with BALB / c were better recipients for the CRM conjugates, than for the N19 conjugates. On the other hand, the CRM conjugates did not promote any antibody response against any of the four polysaccharides in B10.BR H-2k, whereas the N19 conjugates did. Notably, the N19 conjugates promoted substantial antibody responses after the first dose in all strains of mice tested against the four polysaccharides, with few exceptions in the less immunogenic strains. Most of the mice of different genetic backgrounds used in this study produced antibodies to the four polysaccharides, indicating a lack of any apparent genetic restriction of the immune response on immunization with the conjugates of N19. As shown in Figure 14, in accordance with the IgG response measured by ELISA, also the bactericidal titers obtained with the N19 and CRM conjugates were higher in BALB / c H-2d containers. It was observed that the N19 conjugates induced higher bactericidal titers than the CRM conjugates against the four polysaccharides in all tested strains, except in BALB / B mice against MenA. The evaluation of the functional activity of the antibodies produced by bactericidal assay in serum, confirmed in addition the best carrier effect of N19, compared to CRM. 4. Enzyme-linked immunosorbent assay (ELISA) protocols 4.1 Meningococcal polysaccharide specific IgG serogroup A, C, W-135 and Y The titre of MenA-specific immunoglobulin G, MenC, Men and MenY (IgG) was performed on individual sera of each mouse according to the tests already described [297]. 96-well Nuc Maxisorp flat bottom plates were coated overnight at 4 ° C separately with 5 μg / ml of purified polysaccharides from N. meningitidis serogroup A, C, or Y in the presence of 5 μg / ml of methylated human serum albumin . The plates were washed three times with PBS containing 0.33% Brij-35 (PBS-Brij), then saturated with 200 μ? / ???? of PBS containing 5% FCS and 0.33% Brij-35 (PBS-FCS-Brij) for 1 hour at room temperature. Single sera were diluted in PBS-FCS-Brij and titrated against the four polysaccharides separately. The plates were incubated overnight at 4 ° C. The next day, the plates were washed with PBS-Brij, and goat anti-mouse IgG, conjugated to alkaline phosphatase (Sigma Chemical Co., SA Louis, Mo.) Diluted in PBS-FCS-Brij, and the plates were added. were incubated 2 hours at 37 ° C. The bound antibodies were developed using 1 mg / ml of p-nitrophenyl-phosphate (Sigma Chemical Co., SA Louis, Mo.) in diethanolamine solution. After 20 minutes of incubation, the absorbance was read at 405 nm. The pre-immunization values consistently gave an OD value below 0.1. The results were expressed as the titers in relation to a domestic reference serum by parallel line analysis, to minimize the variation from plate to plate. The IgG titers were calculated using the Reference Line Test [298] and expressed as the EU / ml logarithm. 4. 2 Isotype IgGl / IgG2a specific for serogroup A and C meningococcal polysaccharide To measure specific IgG1 and IgG2a antibodies of anti-MenA and anti-MenC, the plates were coated overnight at 4 ° C with 5] iq of methylated human serum albumin / ml and 5 g of menA or MenC purified by me in PBS, as described above for the IgG ELISA. The plates were then washed and blocked with PBS-FCS-Brij for 1 hour at room temperature. The serum samples were diluted in PBS-FCS-Brij through two plates in parallel starting from 1: 100 and incubated for 2 hours at 37 ° C. Goat anti-mouse IgG1 or IgG2a antibodies, conjugated to biotin (Southern Biotechnology Associates, Inc.) were added. After 2 hours of incubation at 37 ° C streptavidin conjugated to horseradish peroxidase (DAKO) was added to the wells, and the plates were incubated for 1 hour at 37 ° C. The plates were developed with the substrate 0-phenylenediamine dihydrochloride (Sigma). Titers were calculated as the reciprocal of the serum dilution at which the OD was 0.5 (450 nM). 4. 3 IgG antibodies specific to N19, TT, HA or CRM, DT The titration of carrier proteins N19, CRM197 and their progenitor proteins, in this case tetanus toxoid (TT), hemophilus influenzae (HA) and toxoid from the Diphtheria (DT) was performed on combined serum as previously described [299, 300]. In summary, plates of 96 wells (Nunc Maxisorp) were coated overnight at 4 ° C with 200 μ? of a PBS solution containing separately 2 g / ml of N19, TT, HA or CRM197 or 5 ug / ml DT antigen. The plates were then washed and blocked with PBS-1% BSA for 1 hour at 37 ° C. Serum samples were diluted in PBS-1% BSA -Tween 20 to 0.05% through the plate starting from 1: 100 and incubated for 2 hours at 37 ° C. For detection, goat anti-mouse IgG, conjugated to alkaline phosphatase, and p-nitrophenyl phosphate were used. The presence of the antigen-specific antibodies was revealed as described above. The results were expressed as the titers relative to a domestic reference serum by parallel line analysis, to minimize plate-to-plate variation. 4. 4 Avidity of IgG antibodies to serogroup A and C eningococci The avidity of group A and C meningococcal IgG antibodies was evaluated by ELISA elution assay of the combined sera using 75 mM ammonium thiocyanate [NH4SCN] as chaotropic agent, according to the well-established method [301, 302]. The validation of the assay included the evaluation of the antigen's ability after incubation with 4 M NH4SCN [303]. Nunc Maxisorp 96-well flat bottom plates were coated all overnight at 4 ° C with 5 pg / ml purified N. meningitidis serogroup A and C polysaccharides, separately. The solution was aspirated and the wells were washed three times with PBS-Brij and blocked for 1 hour at room temperature with blocking buffer (PBS-FCS-Bri j). Plates were washed with wash buffer (PBS-Brij). The test and reference sera were diluted in PBS-FCS-Brij dilution buffer and double serial dilutions in duplicate in a microplate were prepared. After 2 hours of incubation at 37 ° C, the plates were washed 3 times. The serum samples in one of the duplicate were incubated 15 minutes at room temperature with 75 mM NH4SCN in dilution buffer in PBS-FCS-Brij serum, while the other duplicate was incubated with the dilution buffer alone. After washing, the plates were incubated with goat anti-mouse IgG antibodies, conjugated to alkaline phosphatase (Sigma Chemical Co., SA Louis, Mo.) as in the aforementioned ELISA assay. The amount of antibodies remaining bound to the plate after elution with 75 mM NH4SCN was calculated in units of ELISA by reference to the standard ELISA curves, corresponding to 100% bound antibodies. The high avidity IgG titers were represented in percentage of antibodies bound as a function of time.

. Serum bactericidal assay against meningococcal strains A, C, W and Y The method used for the measurement of bactericidal antibody titers has been previously described (94). The target strains N. meningitidis serogroup A (strain F8238), C (strain 11), W (strain 240070) or Y (strain 240539) were grown overnight at 37 ° C with 5% C02 on chocolate agar plates ( starting from a frozen reserve). Colonies with an absorbance of 0.05-0.1 at 600 nm were suspended in 7 ml of ueller Hinton broth containing 0.25% glucose and incubated with shaking for 1.5 hours at 37 ° C with 5% C02 to reach an absorbance of ~ 0.24-0.4 at 600 nm. The suspensions of bacterial cells were diluted in GBSS buffer (Gey's balanced salt solution) (SIGMA) and 1% BSA (assay buffer) to produce approximately 105 colony forming units (CFU) / ml. Combined (50 μ?) Or single heat-inactivated (56 ° C for 30 minutes) serum samples were serially diluted in half (reciprocal initial dilution of 4) in buffer in tissue-treated, flat-bottomed tissue plates , of 96 wells (cost, Inc., Cambridge, Mass). Equal volumes of Cell suspensions and combined baby rabbit supplements (25%) were gently mixed, and 25 μ? to sera diluted in series. The final volume in each well was 50 μ ?. Controls included (i) bacterium-complement-buffer (complement-dependent control) and (ii) serum-bacterium-heat-inactivated test buffer (independent complement control). Immediately after the addition of the baby rabbit complement, 10 μ? Were plated. of the controls on ueller-Hinton agar plates by means of the inclination method (time zero, tO). The microtiter plates were incubated for all serogroup target strains at 37 ° C for 1 hour with 5% C02. After incubation, 10 μ? of each sample were plated on Mueller-Hinton agar plates as dots, while 10 μ? The controls were planted using the tilt method (time one, ti). The agar plates were incubated for 18 hours at 37 ° C with 5% C02, and the colonies corresponding to tO and ti were counted. The colonies to you were a control of the eventual complement or serum toxicity, and it has to be 1.5 times the hills at tO. Bactericidal titers were expressed as the reciprocal serum dilution that produces >50% death compared to the number of target cells present before incubation with serum and complement (tO). The titles were considerable reliable and at least two of the following dilutions produce a bacterial death > 90% Student's t test (2 tails) was used to compare the antibody titers between the groups and at different times. A value P < 0.05 was considered statistically significant. 6. Cell-mediated immune responses 6.1 In vitro proliferation assay with epitopes of N19, NI 9 or NI 9 conjugates of BALBc mice primed with NI 9-menACWY To evaluate immunization with N19 conjugates prepared for carrier-specific epitope T cells , spleens from mice immunized two or three times with N19-MenACWY tetravalent (~6 or 2 pg protein / dose) as described above, were removed 10 days after the last immunization and tested for their ability to proliferate after an in vitro stimulation with simple peptides that constitute free N19 or N19 or conjugated [304]. The purified N19, used in this assay, did not contain detectable LPS, which may have possibly interfered. Spleens from each group of mice were combined and dispersed manually. Once washed and counted, the cells were cultured at a density of 5 x 10 5 cells per well in RPMI (GIBCO BRL Life Technologies) supplemented with 25 mM HEPES buffer, 10 U / ml of penicillin, 100 μg / ml streptomycin, 50 μ-2-mercaptoethanol, 0.15 mM L-glutamine, sodium pyruvate, vitamins, sodium pyruvate and a cocktail of non-essential amino acids (GIBCO BRL Life Technologies 1% of a 100 x stock) ) and 5% fetal calf serum (Hyclone) in 96 well, flat bottom cell culture plates (Corning NY). The cells were cultured in triplicate in the presence of the individual peptides from 0.12 to 30 μ? per well (dilutions two or three times) (~ 0.15-50 g / ml) or free N19 or conjugate from 0.004 to 1 μ? diluted in the same medium, were added to the wells in triplicate to give a total of 200 μ? per well. The controls were run by the complete culture medium or 10 μg / ml of concavalin A, to demonstrate the proliferative capacity of the cells. The plates were incubated at 37 ° C in 5% C02. After five days, the cells were pulsed with 0.5 μ? of [3 H] thymidine (Amersham Biosciences 1 mCi / ml reserve) per well for an additional 18 hours and harvested with the Filtermate Harvester and counted in a liquid scintillation counter (Packard Bioscience). The results of the proliferative assays were expressed as the stimulation index (SI), calculated by the ratio of counts per minute (cpm) in experimental cultures with the stimulus to the per minute counts of control cultures (background) without stimulus. The triplicates of the cultures were run in parallel. A IF > 2 was considered. To determine if the strong effect of N19 helper T cells in the mouse system was mediated by any of the CD4 + epitopes originally included in N19, proliferation of splenocyte T cells from BALB / c mice read two or three times with NI 9-MenACWY (6] sq of N19 / dose) was evaluated. Spleen cells were stimulated in vitro with different concentrations of peptides NI 9 or complete N19, either free or conjugated to polysaccharides. As shown in Figure 15, the lymphocytes proliferated substantially in the presence of free or conjugated N19. T cell proliferation was also observed with the P23TT peptide consistently in all experiments (Figures 15 and 16). Among the other peptides tested, a proliferation of T cells induced by P30TT, P32TT, HA and HBsAg was observed, however only in the presence of higher concentrations. When the tests were carried out with C57BL / 6 mice, none of the epitopes stimulated lymphocyte proliferation and N19 stimulated the cells only at the highest concentration. In addition, the activation of specific N19 T cells in congenic strains of mice was measured to investigate if there was any MHC restriction pattern. Activation was analyzed in vitro by measuring the responses proliferative spleen cells from mice with different genetic backgrounds in the presence of different concentrations of N19, either 1) free or 2) conjugated to polysaccharides, or with 3) peptides that make NI 9 simple or with 4) free polysaccharide components. It was observed that free N19 induced T cell activation in all strains, but N19 conjugates resulted in differential proliferative responses in the strains tested (Figure 17). Assessing the influence of the antecedent genes (BALB or B10) on the responses of H-2, it was observed that the H-2d haplotype mice generated specific T cells for different epitopes. A remembrance of T cells from the P23TT epitope was generated in two genetically unrelated mice (BALB / c H-2d and B10.BR H-2k). On the other hand, congenic mice (BALB or B10) with different H-2 haplotypes, generated different proliferations of T cells specific for the epitope, suggesting that genetic factors outside the MHC complex also influence the response. However, mice with the same genetic background (BALB) generated T cells reactive for the P30TT epitope. In general, despite the fact that the peptides differed in their H-2 restriction level, all strains were able to mount a good antibody response against all four polysaccharides with the N19 conjugates. In addition, it was observed that any of the four polysaccharides were able to induce proliferation in any strain tested, indicating that these are the antigen independent of T cells and the conjugation to a carrier protein does not interfere with its characteristics, such as the ability to induce T cell activation specific of the polysaccharide. 6. 2 Evaluation for the proliferation of murine epitope-specific T cells: immunization protocol and proliferation assay The synthetic peptides (P2TT, P21TT, P23TT, P30TT, P32TT, HA and HBsAg) with a purity of 95% were purchased from Primm s.r.l. (Italy). Groups of three BALB / c mice were immunized subcutaneously at the base of the tail with a volume of 50 μ? per mouse containing 50 iq of a single peptide (P2TT, P30TT, P23TT, P32TT, HA, HBsAg) or N19 emulsified in complete Freund's adjuvant (CFA). 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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (26)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A composition, characterized in that it comprises a combination of two or more monovalent conjugates, wherein each of two or more monovalent conjugates comprise (i) a carrier protein comprising T cell epitopes from two or more pathogens, conjugated to (ii) a saccharide antigen.

2. A multivalent conjugate, characterized in that it comprises two or more antigenic antigens distinct antigens, conjugated to the same carrier protein molecule, wherein the carrier protein comprises T cell epitopes from two or more pathogens.

3. A composition, characterized in that it comprises two or more of the multivalent conjugates according to claim 2.

4. A composition, characterized in that it comprises one or more multivalent conjugates according to claim 2 and one or more monovalent conjugates in accordance with claim 1.

5. The composition in accordance with any of claims 1, 3 or 4, characterized in that the carrier protein in two or more of the conjugates is the same.

6. The composition according to any of claims 1, 3 or 4, characterized in that the carrier protein in each conjugate is the same.

The conjugate according to any of claims 1, 3, 4, 5 or 6, characterized in that at least one of the carrier protein epitopes is not derived from the same pathogen as the saccharide antigen.

8. The conjugate according to any of claims 1 or 3-7, characterized in that none of the epitopes of carrier protein is derived from the same pathogen as the saccharide antigen.

The composition according to any of claims 1 or 4-8, characterized in that a molecule of the carrier protein in the monovalent conjugate is conjugated to more than one molecule of the saccharide antigen.

The composition according to any one of claims 1 or 4-8, characterized in that each carrier protein molecule in each monovalent conjugate is conjugated to more than one saccharide antigen molecule.

11. A conjugate or composition according to any of the previous claims, characterized in that the carrier protein comprises 6 epitopes.

12. The conjugate or composition according to any of the previous claims, characterized in that the carrier protein comprises 19 epitopes.

The conjugate or composition according to any of the previous claims, characterized in that the carrier protein comprises at least one epitope of CD4 + T cells.

The conjugate or composition according to any of the previous claims, characterized in that the carrier protein comprises at least one bacterial epitope and at least one viral epitope.

15. The conjugate or composition according to any of the previous claims, characterized in that at least one epitope of carrier protein is derived from the hepatitis A virus, hepatitis B virus, measles virus, influenza virus, virus of varicella zoster, heat shock proteins of Mycobacterium Bovis and strains of M. leprae and / or Streptococcus.

16. The conjugate or composition according to any of the previous claims, characterized in that at least one of the carrier protein epitopes is selected from tetanus toxin (TT), Plasmodium falciparum CSP (PfCs), nuclear capsid of the virus of the hepatitis B (HBVnc), inina influenza hemagglut (HA), HBV surface antigen (HBsAg) and influenza (MT) matrix.

17. A conjugate or composition according to any of the preceding claims, characterized in that at least one of the carrier protein epitopes is selected from P23TT (SEQ ID NO: 1), P32TT (SEQ ID NO: 2), P21TT (SEQ ID NO. : 3), PfCs. { SEQ ID NO: 4), P30TT (SEQ ID NO: 5), P2TT (SEQ ID NO: 6), HBVnc (SEQ ID NO: 7), HA (SEQ ID NO: 8), HBsAg (SEQ ID NO: 9) ) and MT (SEQ ID NO: 10).

18. The conjugate or composition according to any of the preceding claims, characterized in that at least one of the saccharide antigens is derived from Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Yersinia enterocolitica, Vibrio cholerae, Salmonella typhi, Klebsíella spp., Candida albicans and / or Cryptococcus neoformans.

19. The conjugate or composition according to any of the previous claims, characterized in that it also comprises an adjuvant.

20. The conjugate or composition according to any of the previous claims, characterized in that it also comprises a non-saccharide antigen.

21. The conjugate or composition according to any of the previous claims characterized in that it is for use in therapy.

22. The conjugate or composition according to any of the previous claims characterized in that it is for use in raising an immune response.

23. The use of a conjugate or composition according to any of claims 1-20, in the manufacture of a medicament for producing an immune response in a patient.

24. The use of a conjugate or composition according to any of claims 1-20, in the manufacture of a medicament for producing an immune response in a patient, wherein the patient has been pre-treated with a saccharide antigen different from that comprised within the composition conjugated to a carrier.

25. The use of a conjugate or composition according to any of claims 1-20, in the manufacture of a medicament for producing an immune response in a patient, wherein the patient has been pre-treated with the same saccharide antigen as that comprised within the composition conjugated to a different carrier.

26. A kit, characterized in that it comprises: a) a first conjugate of the invention and b) a second conjugate of the invention.