MXPA00008255A - Multi-oligosaccharide glycoconjugate bacterial meningitis vaccines - Google Patents

Abstract

Multivalent immunogenic molecules comprise a carrier molecule containing at least one functional T-cell epitope and multiple different carbonhydrate fragments each linked to the carrier molecule and each containing at least one functional B-cell epitope. The carrier molecule inparts enhanced immunogenicity to the multiple carbohydrate fragments. The carbohydrate fragments may be capsular oligosaccharide fragments from Streptococcus pneumoniae which may be serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F or 23F), or Neisseria meningitidis, which may be serotype (A, B, C) W-135 or Y. Such oligosaccharide fragments may be sized from about 2 to about 5 kDa. Alternatively, the carbohydrate fragments may be fragments of carbohydrate-based tumor antigens, such as Globo H, LeY or STn. The multivalent molecules may be produced by random conjugation or site-directed conjugation of the carbohydrate fragments to the carrier molecule. The multivalent molecules may be employed in vaccines or in the generation of antibodies for diagnostic applications.

Description

BACTERIAL VACCINES AGAINST GLUCOCONJUGATE-BASED MENINGITIS IN MÜLTIOLIGOSACÁRIDOS FIELD OF THE INVENTION The present invention relates to the field of vaccines and in particular to the field of the development of new glycoconjugation technologies that can be used to prepare glucoconjugates wherein the multioligosaccharides are covalently bound to the same carrier protein.

BACKGROUND OF THE INVENTION Haemophilus influenzae type B (Hib), Neisseria meningi tidis and Streptococcus pneumoniae are the leading causes of bacterial meningitis in children under five years of age. All these bacteria are protected against phagocytosis by a polysaccharide capsule. The antibodies induced against the capsular polysaccharide (CPs) of the organism in the majority of cases are protective. Effective Hib conjugate vaccines where the Hib CP, the PRP, is linked to different carrier proteins, for example diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), CRM 197 (HbOC) and proteins of the outer membrane of N. meningi tidis (HbOC) have already been developed. Currently, four Hib conjugate vaccines are available. The new glucoconjugate vaccines against N. meningi tidis and S. pneumoniae are widely recommended by the College of Physicians of the United States. The development of multivalent pneumococcal vaccines for the prevention of both systemic and non-invasive pneumococcal diseases in infants, geriatric patients, and immunocompromised individuals has gained increasing importance in the last decade. Other detailed reviews of pneumococcal disease, epidemiology or polysaccharide vaccine, and several review articles are available. (reference 1, several references are mentioned in parentheses to describe more fully the state of the art to which they belong.) The complete bibliographic information of each citation is found at the end of the specification, immediately before the claims. incorporated here in this exhibition, as a reference). Streptococcus pneumoniae is a gram-positive, encapsulated bacterium that is present in the normal flora of the human upper respiratory tract. It is a very frequent and important cause of pneumonia, meningitis, bacteremia and noninvasive bacterial otitis media. The incidence of diseases is highest in infants and geriatric patients. In the United States alone, the overall incidence of systemic monococcal infections is estimated at 50 / 100,000 in the geriatric population and 160 / 100,000 in children under 2 years (references 2, 3). Mortality can be up to 40,000 / year, especially in the geriatric population. Many serotypes of S. pneumoniae have developed resistance to conventional antibiotic treatments. The incidence of otitis media in children approaches 90% at the age of five years and presents a peak between six and fifteen months of age. It was estimated that more than 1.2 million cases of otitis media occurred annually. Recent studies in the epidemiology of pneumococcal disease (ref 4) have shown that five serotypes (6B, 14, 19F, 23P and 18C) of the 85 known serotypes produce 70 to 80% of pneumococcal diseases in infants and that in the United States, types 9B and 4 are classified as the fifth and the seventh. In Europe and in developing countries, types 1 and 5 are the most prevalent with respect to types 4 and 9V. Therefore, the pneumococal conjugate vaccine for the United States must contain at least seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) to achieve 75 to 85% coverage. Conjugate vaccine formulations for Europe and elsewhere should include serotypes 1, 5, 6B, 14, 18C, 19F and 23F. A multivalent pneumococal conjugate vaccine, broad spectrum, must contain CP of the nine serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. N. meningi tidis is a gram negative bacterium that has been classified serologically in groups A, B, C, 29e, W135, X, Y and Z. Additional groups (H, I and K) were isolated in China and the group L was isolated in Canada. The grouping system is based on the capsular polysaccharides of the organism. In contrast to the pneumococcal vaccine, the composition of the meningococcal polysaccharide vaccine has been greatly simplified by the fact that much less polysaccharides are required. In fact, groups A, B and C are responsible for approximately 90% of cases of meningococcal meningitis. The prevention of group A and C meningococcal meningitis can be achieved by vaccination with a bivalent polysaccharide vaccine. This commercial vaccine has been used successfully in adults over the last decade to prevent major epidemics of meningitis in many parts of the world. However, there is a need to improve this vaccine because a significant proportion of cases of meningococcal meningitis caused by serotypes other than A and CN meningi tidis of group B is of particular epidemiological importance, but groups Y and W135 are also important . Although the vaccine is currently a tetravalent vaccine comprising the polysaccharides of groups A, C W135 and Y for meningococcal meningitidis, it is not very effective in young children, since the maturation of the immune response to most capsular polysaccharides in infants it occurs at approximately 2 years of age. The meningococcal polysaccharide group B is very little immunogenic in man. The two main reasons for this phenomenon are the following. On the one hand the homopolymer of sialic acid linked to (2- >8) is rapidly depolymerized in human tissue during neuramidase. On the other hand is the capsular polysaccharide group B which is a polymer of N-acetylneuraminic acid (a 2-8 NeuNAc) and that the entity to 2- »8 NeuNac is found as a monomer and a dimer in several glycoproteins and gangliosides in adults and as a polymer with at least 8 repeat units in tissues of newborns and rat fetuses. Therefore, this structure is recognized as a "self" antigen by the human immune system. As a result, the production of the antibody is suppressed or due to this molecular imitation, a vaccine based on the CPs of the native group B could induce autoantibodies directed against the entity at 2-8 NeuNac and therefore produce diseases P1120 autoimmune. As the meningococcal CPs of group B are not immunogenic in humans, approaches have been sought to increase their immunogenicity. One approach uses non-covalent complexes of Group B CPs and outer membrane protein (OMPs). These complexes are formed by the hydrophobic interaction between the hydrophobic regions of the OMPs and the diacylglycerol group at the reducing end of the CPs. Two doses of the complex were administered to human volunteers at 0 and 5 weeks. Most individuals responded with an increase in antibodies to the CPs of group B. However, the second dose resulted in very little increase or zero increase in antibody titers that subsequently declined over a period of 14 weeks. Antibodies with specificity to polysaccharide group B were limited to the IgM class and were directed against determinants present only in high molecular weight polysaccharides. To improve the immunogenicity of the B group CPs, Jennings (ref 5) prepared a conjugate of meningococcal polysaccharide group B-tet toxoid (GBMP-TT) by covalently joining the CPs to the tet toxoid (TT) through its non-reducing cyhalic acid. , terminal using oxidized CPs with periodate. This procedure, however, did not result in a significant improvement in the P1120 immunogenicity of CPs. The antibody response produced in animals was mainly directed against the point of binding between the CPs and the protein (GBMP-TT). Another improvement in the immunogenicity of the B group CPs involved their chemical modification. Jennings (ref 6) reported that the N-acetyl groups of the CPs of group B could selectively be removed by the action of a strong base at elevated temperature. The acetyl groups were then replaced with N-propionyl groups by treatment with propionic anhydride to produce polymers of N-propionylneuraminic acid (a (2-8) NeuPro). The N-propionylated CPs were first oxidized with periodate, sodium periodate, and then coupled with TT in the presence of sodium cyanoborohydride to give the chemically modified conjugate GBMP-TT. Mice immunized with this conjugate formulated in Freund's complete adjuvant (FCA), generated high levels of trans-reactive IgG antibody against native B-group CPs. The murine antisera were found to be bactericidal for all strains group B. However, other studies revealed the existence of two populations of antibodies with different specificity. One population reacted with purified B-group CPs while the other did not. The antibodies that did not react with the CPs of the native group were found to be responsible for the activity P1120 bactericidal. These antibodies can recognize an epitope expressed by CPs associated with cells, which is not present in the purified CP. The alternative conjugates comprising the capsular polysaccharide of the Group B CPs of N-meningi tidis were conjugated with a carrier protein as immunogenic compositions, including vaccines, and their use for the generation of diagnostic reagents, has already been described by Kandil et al. . (U.S. Patent No. 5,780,606, assigned to the assignee of the present and the disclosure of which is incorporated herein by reference). In particular, the capsular polysaccharides of N. meningi tidis contain multiple sialic derivatives that can be modified and used to bind carrier molecules. The dramatic reduction in type b diseases Haemophilus influenzae in countries that have licensed and used conjugate vaccines from Hib-protein CPs, have shown that CPs-protein conjugates can prevent systemic bacterial diseases. It is reasonable to expect that conjugates of meningococcal and pneumococcal-protein CPs will also be effective. The possibility of avoiding non-invasive diseases, for example otitis media, by systemic immunization with conjugate vaccines needs to be explored. When there are high titres of antibodies specific for the serotype, it is sufficient P1120 avoiding either nasopharyngeal colonization and / or otitis media remains an open concern. The development of a vaccine against otitis media requires a multiple pneumococal CPs protein conjugate to produce anti-CPs antibody titers in the first years of life. The development of conjugate vaccines of multivalent-type pneumococal and meningococcal-type CPs, to avoid systemic and non-invasive diseases, presents many challenges for chemists in the area of carbohydrates, for immunologists, for physicians and for manufacturers of vaccines The amount of carbohydrate, the choice of carrier, the method of administration of the vaccine and the use of immunological stimulators or adjuvants, have an influence on host immune responses. Immunogenic glycoconjugates can be formed between proteins and multifunctional CPs if the conditions are controlled very carefully. Most conjugates today are synthesized by coupling reaction, either with CPs or with oligosaccharides activated through the reduction end, to a protein or peptide, with or without a binding group. A general glycoconjugation method involves the random activation of the capsular polysaccharide or fragments of the polysaccharide by treatment with periodate. The P1120 reaction leads to a random oxidative cleavage of the vicinal hydroxyl groups of the carbohydrates with the formation of the reactive aldehyde groups. The coupling to the protein carrier is done by direct amination with the lysyl groups. A spacer group, for example aminocaproic acid, can react with the aldehydes by reductive amination and then copulate with the lysyl groups of protein by water-soluble carbodiimide condensation (ref 7). The oligosaccharide-peptide conjugate reported by Paradiso (ref.8) was similarly prepared except that a peptide having a T cell epitope of CRM? 97 was used in place of the native protein. Another approach to conjugation has been disclosed by Gordon in U.S. Patent No. 4,496,538, assigned to Connaught Laboratories Inc., and by Schneerson et al. (ref 9) which involves the direct derivatization of the CPs with adipic acid dihydrazide (ADH) followed by CNBr activation and then the conjugation of the derivatized CPs, directly with a carrier protein (D or T) by carbodiimide condensation. Marburg and Tolraan (EP # 534764A1) demonstrated that the conjugated immunogens of dimeric protein-CPs can be produced by first coupling the CPs with a protein carrier and then linking the second CPs with the first CPs by a bifunctional trans-linker.

The methods for inducing immunity against the disease are constantly improved. Research has focused on the structure-function relationship of the carbohydrate-protein conjugate with the hope of discovering mechanisms of B and T cell interactions with conjugates that would lead to vaccines with improved immunogenicity and the development of adjuvants and delivery systems. Chong et al. (US Patent No. 5,679,352, assigned to the assignee herein and whose disclosure is incorporated herein by reference) showed that several factors affect the immunogenicity of carbohydrates. The minimum requirements for synthesis of an immunogenic glycoprotein conjugate are that the B cell epitopes of the CPs and the T cell epitopes of the carrier must be functional after covalent attachment. The magnitude of the anti-CPs antibody response depends markedly on the spatial orientation of the CPs relative to the T cell epitopes. The anti-CPs antibody responses improve when multiple antigenic peptides (MAPs) are used as carriers. A single-dose polyvalent vaccine is listed as the first priority in the WHO vaccine development program. The conjugate vaccine of single-dose polyvalent CPs-protein (15 different conjugates of CPs-protein: 1 conjugate Hib, five conjugates N. meningi tidis and nine conjugates S. pneumoniae) against bacterial meningitis, presents a potential risk of hyperimmunization against classical carrier proteins, for example tetanus and diphtheria toxoids. It is documented that nonspecific epitope suppression for the antibody response to Hib conjugate vaccines results in pre-immunization with carrier proteins (ref 11). Therefore, adequate approaches are required to solve this problem of vaccine formulation. Some of the problems can be avoided by incorporating non-capsular, trans-protective and conserved antigens of Hib, N. meningi tidis and S. pneumoniae. Although several outer membrane proteins have been proposed as vaccine candidates, none have been tested in clinical trials. Several multiple-carrier CPs conjugate delivery systems thus represent a novel generic approach and will be important in the development of a glucoconjugate vaccine. Therefore, this invention is directed to new glycoconjugation technologies, which can be used to prepare vaccines containing oligosaccharides from different bacteria covalently linked to the same carrier protein or polypeptide.

SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a multivalent immunogenic molecule, comprising a carrier molecule that contains at least one functional T cell epitope and multiple different fragments of carbohydrate, each linked to the molecule carrier and each contains at least one functional B-cell epitope, wherein the carrier molecule imparts enhanced immunogenicity to the multiple carbohydrate fragments. In one embodiment of the invention, the carbohydrate fragments are fragments of bacterial capsular oligosaccharide. These capsular polysaccharide fragments may be oligosaccharide fragments of Streptococcus pneumoniae, including fragments derived from at least two capsular polysaccharides of S. pneumoniae, serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. The carrier molecule can be a protein containing T cell epitope or a protein fragment of S. pneumoniae. The capsular polysaccharide fragments can be oligosaccharide fragments of Neisseria meningi tidis, including fragments derived from at least two capsular polysaccharides of N. meningi tidis, Groups A, B, C, -135 and Y. The carrier molecule can be a protein containing T cell epitope or a protein fragment of N. meningi tidis. The capsular polysaccharides employed in this aspect of the invention can be oligosaccharide fragments ranging from about 1 to 5 kDa. These fragments can be provided by acid hydrolysis of the respective capsular polysaccharide. The oligosaccharide fragments can be chemically modified to be coupled to the carrier molecule. The carrier molecule can be an oligopeptide containing at least one functional T cell epitope or a carrier protein, for example a tetanus toxoid. In another embodiment of the invention, the carbohydrate fragments are fragments of tumor antigens based on carbohydrate. These carbohydrate-based tumor antigens can be Globe H, Law or STn. According to another aspect of the present invention, there is provided a method for forming a multivalent immunogenic molecule, comprising treating at least two different carbohydrate molecules to obtain carbohydrate fragments thereof and conjugating each of the carbohydrate fragments with a carrier molecule. In one embodiment, the carbohydrate molecule is a capsular polysaccharide of a bacterium and fragments of P1120 oligosaccharide of the capsular polysaccharide that are selected with a size between 2 and 5 kDa. These oligosaccharide fragments are generally derived from at least two different serotypes of the same bacteria, including S. pneumoniae and N. meningi tidis. In this embodiment of the present invention, these multivalent immunogenic molecules can be provided by the glucoconjugation of three or more chemically activated capsular polysaccharides or their derivatives simultaneously with a single carrier molecule, providing a random conjugation. This procedure is illustrated in Figure 1. In this embodiment of the invention, the rational design of lysine-branched peptide systems can be employed for site-directed glycoconjugation. By using different side chain protective products for lysine and cysteine residues during peptide synthesis, the activated oligosaccharides can be selectively and sequentially linked to the same carrier molecule through these residues. This procedure is illustrated in Figure 2. The method of site-directed conjugation may comprise first: forming a peptide of multiple antigen as the carrier molecule and anchored to a polymeric anchor, wherein at least two peptide segments P1120 hauler have different terminal protective groups. One of the protective groups is then selectively removed and first one of the oligosaccharide fragments is coupled to the segment of the unprotected carrier peptide. Another of the protecting groups is selectively removed and a second of the oligosaccharide fragments to the unprotected carrier peptide segment. This procedure can be repeated with the carrier peptides and oligosaccharide fragments that are provided and in relation to which coupling is desired. The resulting molecule is broken from the polymer anchor. According to a further aspect of the invention, there is provided an immunogenic composition for protection against meningitis, comprising: (1) a multiple pneumococcal glucoconjugate according to claim 3, (2) a multiple meningococcal glucoconjugate according to claim 6 and (3) a conjugate of PRP- Immunogenic synthetic peptide. The multiple pneumococcal glucoconjugate can be derived from at least two capsular polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. Multiple meningococcal glucoconjugate can be derived from at least two capsular polysaccharides of N. meningi tidis Groups A, B, C, -135 and Y.

P1120 This universal immunogenic composition of meningitis can be combined with at least one other antigen, for example DTP-polio, to provide a polyvalent vaccine. The present invention further includes a method for generating an immune response in a host, by administering to the host an immunoeffective amount of an immunogenic composition of the present invention. The invention extends to the immunogenic composition claimed herein, when used as a medicament against meningitidis, as well as for the use of the individual component of the immunogenic composition in the manufacture of a medicament against meningitidis. The present invention further includes methods and diagnostic kits using multivalent immunogenic molecules such as those provided herein. Accordingly, in a further aspect of the invention, there is provided a method for determining the presence of antibodies specifically reactive with a multivalent immunogenic molecule, as provided herein, comprising: (a) contacting the sample with the multivalent immunogenic molecule to produce complexes comprising the molecule and any of the antibodies present in the sample, P1120 specifically reactive with it; and (b) determine the production of complexes. In a further aspect of the invention, a diagnostic kit A is provided for determining the presence of a multivalent immunogenic molecule, as provided herein, comprising: (a) The multivalent immunogenic molecule; (b) a means for contacting the multivalent molecule with the sample, to produce complexes comprising the multivalent molecule and any of the antibodies present in the sample; and (c) a means for determining the production of the complexes. Therefore, the present invention allows pneumococal glycopeptide conjugates to be used in a diagnostic immunoassay procedure or diagnostic kit to detect the presence of anti-pneumococal protein and CPs antibodies, eg, anti-CPs 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and anti-pneumococal surface protein antibodies A or anti-meningococcal protein and CPs antibodies, for example anti-CPs A, B, C, Y and -135 and class antibodies 1 anti-meningococal OMP. In a further aspect of the present invention, P1120 provides a method for determining the presence of multivalent immunogenic conjugate molecule in a sample, comprising the steps of: (a) immunizing a subject with an immunogenic conjugate molecule as provided herein, to produce antibodies specific for the carbohydrate fragments; (b) isolating specific antibodies from the carbohydrate fragment; (c) contacting the sample with the isolated antibodies, in order to produce complexes comprising any of the multivalent immunogenic molecules present in the sample and the specific antibodies of the isolated carbohydrate fragment; and (d) determine the production of the complexes. A further aspect of the present invention provides a diagnostic kit for determining the presence of a multivalent immunogenic molecule, as provided herein, in a sample, comprising: (a) the multivalent immunogenic molecule; (b) a means for contacting the multivalent molecule with the sample, to produce complexes comprising the multivalent molecule and any of the antibodies present in the sample; and (c) a means for determining the production of the complexes. A further aspect of the present invention provides a diagnostic kit for determining the presence of a multivalent immunogenic molecule in a sample, comprising: (a) antibodies specific for the carbohydrate fragments of the multivalent immunogenic molecule; (b) means for contacting the antibodies with the sample, to produce complexes comprising multivalent immunogenic molecules and antibodies; and (c) means for determining the production of the complex. The present invention also extends to the use of a mixture of anti-meningococcal CPs antibodies and anti-pneumococcal, anti-PRP CPs, as components in the diagnostic immunoassay kit to detect the presence of Hib, S. pneumoniae and N. meningi tidis in biological samples, for example serum samples.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood from the P1120 following specific descriptions and examples in relation to the accompanying drawings, wherein: Figure 1 shows a schematic diagram of several pneumococcal CPs randomly conjugated with a carrier protein, for example TT, in the procedure employed. Figure 2 shows a schematic diagram of the sequential cross-linking of pneumococcal oligosaccharides chemically activated to a lysine-branched peptide containing several functional T-cell epitopes from pneumococcal proteins. Figure 3 shows the elution profile obtained during the purification of hydrolysed oligosaccharides with S. pneumonaie acid 14, using gel permeation chromatography on a Sephadex®-Gl00 column. Figure 4 shows the elution profile obtained during the purification of hydrolysed oligosaccharides with N. meningi tidis Group B acid using gel permeation chromatography on Sephadex®-Gl00. Figure 5 shows an elution profile obtained during the purification of multivalent oligosaccharide conjugates S. pneumoniae-T. Figure 6 shows rabbit antibody responses to the multivalent conjugates of S. pneumoniae-TT oligosaccharides formulated in FCA.

Figure 7 shows rabbit antibody responses to multivalent oligosaccharide conjugates of S. pneumoniae-TT. Figure 8 shows mouse antibody responses to multivalent conjugates of S. pneumoniae-TT oligosaccharides formulated in FCA. Figure 9 shows rabbit antibody responses to multivalent oligosaccharide conjugates of N. meningi tidis-TT in FCA. Figure 10 shows rabbit antibody responses to multivalent conjugates of S. pneumoniae glycopeptide formulated in FCA. Figure 11 shows rabbit antibody responses to multivalent conjugates of oligosaccharides of S. pneumoniae-WAP, formulated in FCA.

GENERAL DESCRIPTION OF THE INVENTION As discussed above, the present invention relates to novel glycoconjugation technologies that can be used to covalently bind either the multiple oligosaccharides of the bacteria, for example H. influenzae, N. meningi tidis, S. pneumoniae, E. coli and Streptococcus Group B or carbohydrate-based tumor antigens, with the same carrier protein or polypeptide (s) and with the multivalent molecules produced P1120 by the same. The development of resistant and durable humoral immunity requires the recognition of foreign antigens by at least two separate subsets of lymphocytes. B lymphocytes (B cells, lymphocytes derived from bone marrow), are the precursors of the antibody-forming cells and T lymphocytes (T cells), lymphocytes derived from the thymus) modulate the function of B cells. Most CPs they are T-cell independent antigens and are able to directly stimulate B cells to produce antibodies. In general, CPs induce B cells to terminally differentiate them within the antibody-secreting cells (plasma cells), but the antibody responses are short-lived and are limited by the number of responding B cells. Proteins and peptides are T cell-dependent antigens and contain epitopes that can form class II peptide: MHC complexes on a B cell and activate T helper cells to synthesize cytokines linked to secreted cells and cytokines (effector molecules) that synergize in B cell activation and clonal expansion. CPs can become T-dependent antigens by coupling with a protein Carrier P1120 or T cell epitope (ref 9; U.S. Patent No. 4,496,538). Through repeated immunization with CPs-protein conjugates, the B cell population in the vaccines enters not only the production of antibodies, but also the proliferation and maturation. As a result, there are more B cells that produce anti-CP antibodies and there are higher antibody titers in response to the boosters.

Justification for the use of oligosaccharides as antigens The minimum requirements for producing conjugates of immunogenic glycoprotein are that the B cell epitopes of the CPs and the T cell epitopes of the carrier are functional after covalent attachment. To randomly conjugate two or more CPs to the same carrier protein or T cell epitopes, the size of the carbohydrate is reduced to approximately 2 kDa to 5 kDa to avoid spherical hindrance effects. At least two different approaches may be used to covalently link multiple oligosaccharides to a carrier protein. The first approach is to activate or derivatize the oligosaccharides using the same chemistry, so that their conjugation to the carrier can be achieved simultaneously (Figure 1). The second approach uses peptide systems P1120 branched with lysine for site-directed glycoconjugation. The use of different side chain protective groups for cysteine and lysine residues during peptide synthesis makes the activated oligosaccharides can be selectively and sequentially coupled to the same carrier protein by these residues (Figure 2).

Preparation of oligosaccharides As described in detail in the following examples, acid hydrolysis of different serotypes of capsular polysaccharides of Streptococcus pneumoniae (> 50 kDa) can be carried out to form oligosaccharides with molecular weights ranging from about 2 to 5 kDa . This process may comprise three steps: (1) acid hydrolysis of CPs in a sealed vial under an argon or other suitable inert gas atmosphere; (2) lyophilization and (3) purification of oligosaccharides by gel filtration chromatography. The protocol for the acid hydrolysis of the CPs of S. pneumoniae serotypes 1, 4, 5, 9V and 14 has been optimized. Typically, the CPs (2 mg / mL) are incubated in 0.5 M trifluoroacetic acid (TFA) at about 60 ° C to 90 ° C for about 5 to 10 hours. As the CPs of serotypes 6B and 19F contain links of P1120 labile phosphodiester, its hydrolysis is carried out under mild acid conditions (in acetic acid of between 10 and 50 mM, approximately) and between approximately 50 ° and 100 ° C for approximately 30 to 48 hours. CPs of serotype 23F can be partially hydrolyzed either by incubating in approximately 0.1 to 0.5 M trifluoroacetic acid (TFA) at about 70 ° C for about 2 to 4 hours, or in acetic acid of about 1 to 50 mM, between about 80 and 110 ° C, for approximately 40 to 60 hours. At the end of each hydrolysis, the reaction solutions are diluted 5 times with water, then lyophilized. Purification of the crude oligosaccharides can be achieved using gel filtration chromatography with Sephadex® G-100 (approximately 2 x 210 cm column) or any other convenient gel filtration column. Typical chromatography results are illustrated in Figure 3. Fractions are titrated for presence of carbohydrate using resorcinol / sulfuric acid assay (ref 12). The elution profile is plotted and the chromatographically purified oligosaccharides with an average mass of approximately 2 to 5 kDa are pooled. Molecular weight markers are used to calibrate the column, as follows. Patterns of dextran (39,100 and 8,800 Da), PRP hexamer Synthetic P1120 (2,340 Da), sucrose (342 Da) and glucose (180 Da). The oligosaccharides are sized between about 2 and 5 kDa containing about 4 to 8 repeating units, in general, and are expected to contain at least one B-cell epitope. The yields of these oligosaccharides are from about 70 to 90%. These chromatographically purified oligosaccharides are then used to prepare glycoconjugates comprised of multiple oligosaccharides covalently linked to a carrier protein or to a multiple antigen peptide (MAP) system containing T cell epitopes derived from S. pneumococcal proteins. As described in detail in the following examples, the acid hydrolysis of various serotypes of capsular polysaccharides of N. meningi tidis (>; 10 kDa) can be carried out to form oligosaccharides with an average molecular weight of about 2 to 5 kDa. In common with the acid hydrolysis of pneumococcal CPs, the process comprises acid hydrolysis, lyophilization and purification using gel filtration chromatography. The conditions of the acid hydrolysis of the CPs of N. meningi tidis group C, W-135 and Y have been optimized. Typically, the CPs (10 mg / mL) were mixed with approximately 20 to 80 mM sodium acetate, pH from about 4.5 to 5.5, in several vials under atmosphere P1120 of argon or any other suitable inert gas, between approximately 65 to 100 ° C, for approximately 8 to 12 hours. As the CPs of group B can undergo intramolecular esterification under acidic conditions, the conditions used for the hydrolysis of the CPs group C are those used, but the incubation time is limited between approximately 1 hour and the pH of the reaction is immediately adjusted to pH 7, with approximately 0.1 M NaOH, to reverse the esterification process. The group A CPs contain labile phosphodiester bonds, therefore they are hydrolyzed under mild acidic conditions (for example with acetic acid of between about 10 and 20 mM) and incubated at a temperature between about 50 and 100 ° C for about 30 a 48 hours. At the end of each hydrolysis, the reaction solutions are diluted 5 times with water and then lyophilized. The crude oligosaccharides are fractionated by Sephadex® G-100 gel filtration chromatography (column of approximately 2 x 210 cm, as above). Typical results of chromatography are illustrated in Figure 4. Fractions are titrated for presence of cyhalic acid using resorcinol / sulfuric acid titration (ref 12). The elution profile was plotted and the chromatographically purified oligosaccharides between about 2 and 5 kDa were P1120 meet. Dimensioned oligosaccharides of about 2 to 5 kDa typically contain between about 6 and 15 repeating units and are expected to contain at least one B cell epitope. The yields of these oligosaccharides are between about 40 and 80%. These chromatographically purified oligosaccharides are used to prepare glycoconjugates comprised of multiple oligosaccharides covalently linked to a carrier protein or to a multiple antigen peptide (MAP) system containing T cell epitopes from meningococcal proteins. Similar procedures can be used for capsular polysaccharides of other bacteria.

Selection of the carrier or carrier Although several meningococcal and pneumococcal membrane proteins have been identified as potential protective antigens, such as pneumolysin (ref 13), surface protein A. pneumococal (PspA) (ref 14), 37 kDa S. pneumoniae protein (SP37) (ref 15), meningococcal transferrin binding protein 2 (Tbp2) (ref 16), meningococcal pilin (ref 17) and class 1 proteins (ref.18), none of them to date has been tested in clinical trials. These P1120 proteins contain potential T cell epitopes that have been identified using conventional algorithms. Therefore, a panel of potential peptide carriers may be selected for conjugation with the meningococcal and pneumococcal oligosaccharides to form the multivalent immunogenic molecules herein. In the present invention, the peptides (Table I; SEQ ID NOS: 1 to 8) which are to be coupled to the oligosaccharides were selected on the basis of their potential T cell stimulator properties or their potential protective ability or for the conservation of sequences that could be important for remembering the memory of the T cell. NMTBP2 (SEQ ID NO: 1) is a peptide fragment of the Tbp2 protein of N. meningi tidis and has previously been identified as containing functional T cell epitopes as well as which strain-specific protective B-cell epitopes recognized by a specific Tbp2 MAb (U.S. Patent No. 5,708,149 assigned to the assignee herein and whose disclosure is incorporated herein by reference, WO95 / 13370). Peptides NMC-1 and -2 (SEQ ID NOS: 2 and 3) were identified to contain the immunodominant human T cell epitopes of the class 1 protein of N. meningi tidis (ref 19). ? MPi-1 (SEQ ID? O: 4) was derived from N. meningi tidis pilin protein and was P1120 shows that they contain sequences involved in the adhesion (ref 17). The PN peptides (123 -140); SEQ ID NO: 5) and (PN (263-281; SEQ ID NO: 6) pneumolysin derivatives of S. pneumoniae, both contain functional T cell epitopes (ref 20) SP37 (SEQ ID NO: 7) is the N-terminal fragment of the 37 kDa S. pneumoniae protein and shown to be highly immunogenic in rabbit immunogenicity studies PSP-AA (SEQ ID NO: 8) is the N-terminal fragment of the S protein. pneumoniae PspA and shown to be able to produce protective immunological responses in mice, against bacterial pneumococal inoculation (ref 14).

Immunogenicity of multioligosaccharide-carrier conjugates in animal models A. Random conjugation approach (Figure 1) In the present invention, acids have been used to hydrolyse bacterial capsular polysaccharides in low molecular weight oligosaccharide fragments. The oligosaccharides can be purified and reacted with ammonia or with diaminoethane to generate a free amino terminal group at their reducing ends. The amino groups are then reacted with an excess of disuccinimidyl ester of adipic acid to introduce an active succinimidyl ester group into the oligosaccharides. The P1120 activated oligosaccharides are then reacted with amino groups of carrier or peptide proteins to form covalent amide bonds. The glucoconjugates comprise at least two coupled oligosaccharides per protein / peptide molecule. To avoid anti-binding antibody responses, the oligosaccharides can be coupled directly to the carriers using the reductive amination procedure described by Jenning and Lugo sky (ref 6). The advantages of this last procedure are that the linker molecules are not necessary, which eliminates the formation of potential neoantigen groups, and also that very stable secondary amine is formed or, in some cases, a tertiary amine bond, between the oligosaccharide and protein. Furthermore, the treatment of most of the meningococcal and pneumococcal capsular polysaccharides with periodate does not produce a reduction in the molecular weight of the polysaccharide or fragment, since the oxidation is carried out either in the branching side chains or in the residues of cyclic sugar of the main chain. In any case, the main chain does not break and the molecular size remains intact. To evaluate the potential use of the multivalent molecules of the present invention, the oligosaccharides of S. pneumoniae serotypes 6B, 14, 19F and 23F were linked P1120 np randomly and covalently with TT, as shown in Fig. 1. The resulting multiantigen gluaoconjugate (PIAH) was purified by a & amp; filtration in gßl (Figure 5). The analysis of proteins and carbohydrates revealed that the carbohydrate to protein molar ratio was 7.3. :1. Four individual conjugates < S-TT, 14-TT, i 9F-TT and 23F-TT) were prepared, starting with fragments of the respective four serotyps with the same method for comparative studies, multiple antigenic glusoconjugate (MAG) was formulated with adjuvant Complete Freund (FCA) or with alum. The results of immunogenicity studies in rabbit and mouse (BALB / c) indicated that i a. Strong antibody responses were observed for the four serotypes of CPs in rabbits, when using 1 < 'CA as adjuvant (Figure G). The titles were comparable to those obtained with individual conjugates. b. When alum was used as an adjuvant in rabbits, only anti-14, anti-19F and anti-23F antibody responses were observed and no anti-6B responses were found (Figure 7). c. Only anti-14 and anti-19F antibodies were produced in BALB / c mice (Figure 8).

P1-.20 The biological activity of the anti-pneumococcal antibodies was assessed by two different methods: in vitro opsonophagocyte assays and animal protection studies in vivo using active or passive immunization. Previous studies have been shown (refs. 21 and 22) in which the anti-CPs antibodies of S. pneumococcal were biologically active and protective. There was a direct correlation between the ELISA titers of total Ig antibody and the opsonization titers. Therefore, the candidate pneumococcal MAG vaccine that can produce anti-CPs antibody responses of S. pneumoniae in animal models will be useful for human immunization. A glucoconjugate of N. meningi tidis containing oligosaccharides of group C, W and Y was prepared as described above following the procedure shown schematically in Figure 1. The multiple antigen glycoconjugate was purified by gel filtration chromatography. The molar ratio of carbohydrate to protein was 6.6: 1. Rabbit immunogenicity studies revealed that meningococcal MAG could produce antibody responses against all three polysaccharides (C groups), and Y) in carbohydrate-specific ELISAs (Figure 9) and that the antisera had no reactivity against S. pneumoniae GB PC that was used as a negative control. The reactivities of antibodies against P1120 groups W and Y were very similar (geometric mean of the title (GMT) approximately 3000). Group C was less immunogenic in this multivalent glycoconjugate with a GMT of about 500. B. Multiple antigenic peptide (MAP) approach (Figure 2) In this invention, we provide methods for designing and synthesizing novel lysine branching peptides containing different auxiliary cell epitopes T (multiple antigenic peptide, MAP) to which several different oligosaccharides can be coupled selectively and sequentially. To approve this concept, MAP bound to the resin was synthesized and characterized as shown below. Step 1 1-MeOH / ib Step 2 Í-Strcp. pneum -CHO 2-NaCNBHj P1120 An Fmoc-Lys (t-Boc) -TGA resin (500 mg, purchased from DACHEM) with a substitution level of 180 μmol / g was used to prepare MAP. A standard Fmoc chemical coupling protocol was the one used (4 times excess of Fmoc-protected amino acids, 0-benzotriazoyl-N, N, N-N-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBT) / diisopropylethylamine (DIEA) for 1 hour (example 6)). In order to facilitate the conjugation of oligosaccharides, the level of MAP substitution was reduced to approximately 50 μmol / g when the first Fmoc-Gly residue was coupled. When the synthesis was complete, a small portion of MAP-resin was cleaved with 95% trifluoroacetic acid (TFA) in the presence of ethane dithiol (EDT) and thioanisole. The amino acid analysis revealed that the cleaved MAP did not have the correct amino acid composition. The MAP (150 mg) was treated with dithioanisole (DTT) in dimethyl formamide (DMF) to remove the triphenyl group from the cysteine residues in order to conjugate the derivatized oligosaccharides with the SH-directed functional groups, such as for example m-maleimidobenzoyl -N-hydroxysuccinimide (MBS). After reduction, the resulting MAP resins were titrated to determine amino groups and sulfhydryl groups. From Ellam's assessment, P1120 substitution level SH was 64 μmol per g of MAP resin and the degree of substitution of amino groups was 71 μmol / g from the ninhydrin titration. These results indicated that the Fmoc and trityl protection groups can be removed quantitatively. (PRP) 6-MBS which was prepared from a synthetic hexamer of 3-β-D-ribose- (1-1) -D-ribitol-5-phosphate derivatized with MBS (ref 23) was dissolved in DBF / solution PBS and then coupled to the MAP totally deprotected and reduced, under degassed conditions. After coupling, the MAP- (PRP) 6 was subjected to the Ellman test for the determination of the sulfhydryl group. The level of substitution of SH was reduced to 6.85 μmol per g of resin. The coupling of PRP was independently confirmed by ribose titration and was 18 μg of (PRP) 6 / mg resin. The resulting MAP- (PRP) 6 was mixed with S. pneumoniae serotype 19F, oxidized with periodate (1 eq.) In methanol / phosphate er (pH 7.8) in the presence of NaCNBH3 at 38 ° C for 6 days. After conjugation, the substitution of the amino group determined by the ninhydrin titration was 16 μmol per g of resin. The total sugar content was again 16.1 mg / g resin. A small portion of glycoconjugate 19F- (PRP) 6-MAP-resin was cleaved with 95% TFA in the presence of ethane P1120 dithiol (EDT) and thioanisole. After working, the MAP-glucoconjugate had the correct composition of amino acids and carbohydrate content. These results suggest, in a confirmed manner, that different oligosaccharides can be conjugated selectively and sequentially to the MAP resin. Before synthesizing a MAP resin containing different T-cell epitopes, the pneumococcal oligosaccharides oxidized with periodate from serotypes 6B, 14 and 23F were tested for efficient coupling to the linear peptides bound to resin corresponding to PN (123-140) (SEQ ID NO: 5) or PN (263-281) (SEQ ID NO: 6), or to PN (263-281) (SEQ ID NO: 6), which are T cell epitopes derived from pneumolysin, membrane protein of S. pneumoniae. The linear glycopeptides 6B-PN- (123-140), 14 -PN (263-281) and 23F-PN (123-140) were prepared using reductive amination. The coupling efficiency of the oligosaccharides to the peptide bound to the resin was from 10 to 30% according to the determination of the free amino group using the ninhydrin test. The glycopeptides were cleaved from the resin using 95% TFA, then semi-purified by RP-HPLC. The immunogenicity studies in rabbits were carried out with an "equimolar" combination of these glycopeptides formulated in FCA / IFA. The results P1120 indicated that the glycopeptide conjugates were immunogenic and produced responses of anti-polysaccharide antibodies 6B, anti-polysaccharide 14 and anti-polysaccharide 23F (Figure 10). In addition, the rabbit antisera reacted with the peptides as determined by the peptide-specific ELISA assay (Table 2). A MAP resin containing three T cell epitopes derived from different S. pneumococcal membrane proteins was synthesized using a Fmoc-Gly-Lys-TGA resin with a substitution level of 50 μmol / g, as shown in Figure 2. The complete synthesis was carried out manually using the optimized Fmoc chemical coupling protocol, described above. When the synthesis was complete, a small portion of MAP-resin was cleaved with 95% TFA in the presence of EDT and thioanisole, the cleaved MAP had the correct amino acid compositions by amino acid analysis. The MAP-resin was reduced with DTT to remove the t-butylthio protecting groups from the cysteine residues. After an excess wash, the MAP resin was resuspended in a DMF / PBS solution, then mixed with a 4-fold excess of oligosaccharides activated with sulfosuccinimidyl (4-iodoacetyl) amino benzoate (sulfo-SIAB) of S. pneumoniae serotype 14 (Osl4). After mixing overnight at room temperature, the P1120 MAP resin was collected by filtration and washed with PBS, DMF and then methanol. The MAP-Osl4 resin was subjected to the Ellman test and the sulfhydryl group determination. The substitution level of SH was half the initial value. The re-coupling did not increase the amount of Osl4 conjugated to the MAP resin. The presence of N-acetylgalactosamine (GlcNac) in the glyco-MAP resin, a carbohydrate found in 0sl4, was independently confirmed by carbohydrate analysis. MAP-OS14 was first treated with 1% TFA to remove Mtt (a lysine protecting group) from Mtt-lysine residues, then neutralized with a soft base, 1% diisopropylethylamine (DIEA) / DMF. The presence of free amino groups was assessed by the ninhydrin test which indicated that more than 90% of the Mtt groups had been removed. The MAP-OS14 resin was resuspended in PBS and then mixed with four equivalents of oligosaccharides of S. pneumoniae serotype 6B (0s6B) oxidized by periodate, in a buffer solution of DMF / phosphate (pH 7.8) in the presence of 38 ° NaCNBH3. C for 6 days. After conjugation, the substitution of the "amino" groups was determined by the ninhydrin titration, which was 80 to 90% of the original value.

P1120 coupling did not improve the conjugation of 0s6B to the resin MAP-0sl4. Although the coupling efficiency was low (approximately 15%), the presence of ribitol in the MAP conjugate, a carbohydrate found in 0s6B, was confirmed by carbohydrate analysis. The MAP-Osl4-Os6B conjugate was treated with 20% piperidine in DMF to remove the Fmoc protecting group from the Fmoc-lysine residues. After washing, the MAP-Osl4-Os6B resin was mixed with a 4-fold excess of S. pneumoniae serotype 19F oligosaccharides, oxidized with periodate (Osl9F) in DMF / phosphate buffer (pH 7.8) in the presence of NaCNBH3 at 38 ° C for 6 days. After conjugation, the degree of substitution of amino groups was measured by ninhydrin titration which was 90%. The coupling reaction was repeated and its efficacy was determined to be about 15%. However, the presence of N-acetylmanose (ManNAc), a sugar found in Osl9F, was detected by carbohydrate analysis. A small portion of MAP-resin glycoconjugate was cleaved with 95% TFA in the presence of EDT and thioanisole. After working, the MAP-glucoconjugate was evaluated to determine amino acid composition and carbohydrate content, which were correct. Although the overall performance was very low (approximately 5%), these results show that P1120 can selectively and sequentially conjugate different oligosaccharides to the MAP resin. In addition, immunogenicity studies in rabbits indicate that this MAP glycoprotein conjugate was immunogenic and produced strong antibody responses against 19F and 14 polysaccharides (GMT of approximately 3000), but very weak with anti-6B IgG responses (Figure 11). The antibody titers against polysaccharides 19F and 14 were significantly lower than those obtained in rabbits immunized with multivalent oligosaccharides conjugated to TT (Figure 7), but we still expect that the vaccine candidate of the MAP conjugate, multivalent, pneumococal, is useful for human immunization . In addition, rabbit antisera reacted strongly with T cell peptides in peptide-specific ELISA assays (Table 3).

Utility of the Synthetic Glycopeptide Conjugation Technology In the preferred embodiments of the present invention, the glucoconjugate technology can be used in general to prepare conjugate vaccines against encapsulated pathogenic bacteria. Therefore, the glucoconjugate technology of the present invention can be applied to vaccines to confer protection against infections with any bacterium that expresses antigens of P1120 protective polysaccharide potentials, including Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Neisseria meningi tidis, Salmonella typhi, Streptococcus mutans, Crytococcus neoformans, Klebsiella, Staphylococcus aureus and Pseudomonas aerogenosa. In particular embodiments, the synthetic glucoconjugate technology can be applied to produce vaccines that produce antibodies against proteins and oligosaccharides, including fragments of carbohydrate-based tumor antigens, for example Globo H, Le? and STn. These vaccines can be used, for example, to induce immunity against tumor cells or to produce anti-tumor antibodies that can be conjugated with bioactive or chemotherapeutic agents. It is also understood that within the scope of the invention there are variants or functional equivalents of the above specific peptides. The term "variant" or "functional equivalent variant" as used herein refers to whether the peptide is modified by addition, deletion or derivatization of one or more of the amino acid residues, in any aspect, and still acts in a similar manner. to the specific peptides described herein, then that modified peptide falls within the scope of the invention. Given the amino acid sequence of these peptides (Table 1) and any similar peptide, these are P1120 synthesize easily using commercially available peptide synthesizers, for example Applied Biosystems Model 43OA or they can be produced by recombinant DNA technology. It is evident to those skilled in the art that the various embodiments of this invention have many applications in the field of vaccination, diagnosis and treatment of infections and the generation of immunological reagents. An additional non-limiting analysis of these will be presented below.

Preparation and use of vaccines As already indicated, this invention, in one embodiment, provides useful multivalent immunogenic conjugates for formulating immunogenic compositions suitable for use as, for example, vaccines. The immunogenic composition produces an immunological response by the host to which it is administered, including the production of antibodies by the host. The immunogenic compositions can be prepared in injectable form, as liquid solutions or emulsions. Immunogenic compositions and antigens can be mixed with physiologically acceptable carriers that are compatible therewith. These include water, saline, dextrose, glycerol, ethanol and P1120 combinations thereof. The vaccine may also contain auxiliary substances, for example wetting or emulsifying agents or pH regulating agents, to further strengthen their effectiveness. The vaccines can be administered by subcutaneous or intramuscular injection. Alternatively, the immunogenic compositions formed according to the present invention can be formulated and administered so as to evoke an immunological response on mucosal surfaces. Therefore, the immunogenic composition can be administered to mucosal surfaces, for example, nasally or orally (intragastric). Alternatively, other forms of administration, including suppositories, may be desired. For suppositories, binders and carriers can be included, for example polyalkylene glycols and triglycerides. Oral formulations may include the excipients that are normally used, for example saccharin, cellulose and magnesium carbonate pharmaceutical grade. These compositions may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and may contain between 1 and 95% of the immunogenic compositions of this invention. The immunogenic compositions are administered in a manner compatible with the dose formulation and in a manner P1120 that the amount is therapeutically effective, protective and immunogenic. The amount to be administered depends on the subject to be immunized, including, for example, the ability of the subject's immune system to synthesize antibodies and, if necessary, to produce the cell-mediated immune response. The precise amounts of the immunogenic composition and the antigen to be administered depends on the judgment of the physician. However, suitable dose ranges can be readily determined by those skilled in the art and can be in the order of micrograms to milligrams. Suitable regimens of initial administration and booster doses are variable, but may include an initial administration followed by subsequent administrations. The dose of the vaccine may also depend on the route of administration and will vary according to the size of the host. The concentration of the antigen in an immunogenic composition according to the invention is generally from about 1 to 95%. A vaccine containing antigenic material from only one pathogen is a monovalent vaccine. Vaccines containing antigenic material of various pathogens are combined vaccines and also belong to the present invention. These combined vaccines contain, for example, various material P1120 pathogens or from several strains of the same pathogen, or from a combination of several pathogens. Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants, which are commonly used in a solution of 0.005 to 0.5 percent in phosphate buffered saline. The adjuvants improve the immunogenicity of an antigen but are not necessarily themselves immunogenic. The adjuvants can act by retaining the antigen locally, near the site of administration, to produce a deposition effect that facilitates a slow and sustained release of the antigen to the cells of the immune system. The adjuvants can also attract cells of the immune system to a deposit of antigens and stimulate these cells to produce an immune response. Immunostimulatory agents or adjuvants have been used for many years to improve the immune responses of hosts to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, are usually the components of the attenuated or killed bacteria that are used in vaccines. The extrinsic adjuvants are immunomodulators that are typically non-covalently bound to the antigens and formulated to improve the responses P1120 host immunological. Therefore, it has been identified that adjuvants can improve the immune response of antigens delivered parenterally. Some of these adjuvants are toxic, however, they can cause undesirable side effects, making them unsuitable for use in humans and in many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively referred to as alum) are routinely used as adjuvants in veterinary and human vaccines. The efficacy of alum to increase antibody responses to diphtheria and tetanus toxoids is well established and, more recently, an HBsAg vaccine has used alum adjuvant. While the utility of alum is well established for many applications, it has its limitations. For example, alum is ineffective for influenza vaccination and inconsistently produces a cell-mediated immune response. The antibodies produced by antigens with alum adjuvants are mainly of IgGl isotype in the mouse, which is not optimal for protection with these vaccine agents. A wide range of extrinsic adjuvants can elicit potent immune responses to antigens. These include complexed saponins P1120 to membrane protein antigens (immune stimulatory complexes), pluronic polymers with mineral oil, mycobacteria killed in mineral oil, complete Freund's adjuvant, bacterial products, for example muramyl dipeptide (MDP) and lipopolysaccharide (LPS) as well as lipid A and liposomes. To effectively induce humoral immune response (HIR) and cell-mediated immunity (CMI) normally the immunogens are emulsified in adjuvants. Many adjuvants are toxic, induce granulomas, acute and chronic inflammation (Freund's complete addendum, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although this FCA is an excellent adjuvant and is widely used in research, it does not have authorization for use in human or veterinary vaccines due to its toxicity. The desirable characteristics of the ideal adjuvants include: (1) absence of toxicity; (2) ability to stimulate a long-lasting immune response; (3) manufacturing simplicity and long-term storage stability; (4) ability to produce CMI and HIR for P1120 antigens administered by several routes of administration; (5) synergy with other adjuvants; (6) ability to selectively interact with antigen presenting cell populations (APC); (7) ability to specifically produce specific TH2 or T.l cell-specific immunological responses; and (8) ability to selectively raise the appropriate levels of antibody isotype (e.g., IgA) against antigens. U.S. Patent No. 4,855,283, issued to Lockhoff et al. on August 8, 1989, which is incorporated herein by reference, shows analogues of glycolipids, among which are included N-glycosylamides, N-glucosylureas and N-glucosylcarbamates, each of which is substituted in the sugar residue by a amino acid, as immunomodulators or adjuvants. Therefore, Lockhoff et al. (U.S. Patent No. 4,855,283 and ref 29) reported that N-glycolipid analogues that exhibit structural similarities to natural glycolipids, e.g. glycosphingolipids and glucoglycerolipids, are capable of producing strong immunological responses in herpes virus vaccines simplex and in rabies virus vaccines. Some glycolipids have been synthesized from alkylamines Long chain P1120 and fatty acids that are directly linked with sugars through the anomeric carbon atom, to mimic the functions of natural lipid residues. U.S. Patent No. 4,258,029, issued to Moloney, assigned to the assignee hereof and incorporated herein by reference, shows that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and with Poliovirus vaccines I, II and III, inactivated with formalin. Also, Nixon-George et al. (ref 30), reported that octodecyl esters of aromatic amino acids complexed with a recombinant surface antigen of hepatitis B, improved the immune response of the host against the hepatitis B virus.

Immunoassays In one embodiment, the conjugates of the present invention are useful for the production of immunogenic compositions that can be used to generate antigen-specific antibodies that are useful in the specific identification of that antigen in an immunoassay, according to a diagnostic modality. These immunoassays include assays (ELISA), RIA and other antibody binding assays not linked to enzymes or P1120 procedures known in the art. In ELISA assays, antigen-specific antibodies are immobilized on a selected surface, for example, the cavities of a polystyrene microtiter plate. After washing to remove incompletely absorbed antibodies, a non-specific protein, for example a solution of bovine serum albumin (BSA) or casein, which is known to be antigenically neutral with respect to the test sample, can be ligated to the selected surface. This allows the blocking of non-specific absorption sites on the immobilization surface and therefore reduces the background properties produced by non-specific binding of the antigens on the surface. The immobilization surface is then contacted with a sample, for example with clinical or biological material, which is to be tested, so that an immune complex (antigen / antibody) is produced. This may include diluting the sample with diluents, for example BSA, bovine gamma globulin (BGG) and / or phosphate buffered saline (PBS) / Tween. The sample is allowed to incubate for 2 to 4 hours at temperatures of the order of about 25 ° to 37 ° C. After incubation, the surface in contact with the sample is washed to remove nonimmunocomplexed material. The washing procedure can P1120 include washing with a solution, for example PBS / Tween or a borate buffer solution. After the formation of the specific immunocomplexes between the antigen and the test sample and the antigen-specific antibodies already bound, a subsequent wash is made and after the appearance and even the amount of the immunocomplex formation can be determined by subjecting it to a second antibody having specificity for the antigen. To provide a detection means, the second antibody can have an associated activity, for example an enzymatic activity, which will generate, for example, a color development during incubation with a suitable chromogenic substance. The quantification can then be achieved by measuring the degree of color generation with the use of, for example, a visible spectrum spectrophotometer. In a further embodiment, the present invention includes a diagnostic kit comprising antigen-specific antibodies generated by immunization of a host with immunogenic compositions produced according to the present invention. It is understood that the application of the methodology of the present invention is within the abilities of those who have ordinary skill in the art. The examples of the products of the present invention and the P1120 processes for its preparation and use appear in the following examples.

EXAMPLES The above discussion generally describes the present invention. To understand more fully how it can be obtained, reference will be made to the following specific examples. These examples are described only for the purpose of illustrating the invention and are not intended to limit the scope of the invention. Changes in the constitution of equivalents are considered as circumstances that may be the result of a suggestion or simplicity of the method. Although specific terms have been used herein, those terms are intended to be descriptive and in no way restrictive. Immunological methods are probably not explicitly described in this disclosure, but they are well known within the scope of those skilled in the art.

Example 1 This example shows the preparation of group B meningococcal oligosaccharides (GBM) hydrolyzed with acid. This example describes a method for preparing GBM oligosaccharides (P.M. 3000 to 4500 Da) from P1120 commercial type GBM polysaccharides (P.M. > 10 kDa). Required reagents: 1 - GBM polysaccharides from the Sigma catalog # C-5762 2 - Sodium acetate buffer solution (50 mM) pH 5.00 prepared by mixing a volume of sodium sodium acetate 0.5 M, with a volume of acetic acid 0.23 M. 3 - . 3 - Reaction flask and a magnetic bar for agitation. 4 - Sephadex G-25 gel column 5 - Ammonium bicarbonate (20 mM). Procedure The polysaccharide GBM (200 mg) was dissolved in 15 mL of degassed 50 mM sodium acetate buffer, pH 5.0 and the mixture was then stirred at 80 ° C for one hour. The reaction mixture was then immediately cooled with ice and neutralized to a pH of 7.0 by dropwise addition of NaOH.

The whole mixture was lyophilized to give a crude product (460 mg, containing sodium acetate). Approximately 100 mg of GBM treated with acid are first dissolved in 3 mL of 20 mM ammonium bicarbonate and then loaded onto a Sephadex G-25 gel column equilibrated with 20 mM ammonium bicarbonate solution, using the following conditions: Column: (10 x 1000 mm), calibrated with dextran 8800, β-cyclodextran and sucrose standards.

P1120 Flow rate: 0.6 mL / min. Regulator: 20 mM ammonium bicarbonate Fractions collected at 4.5 min / tube. The fractions were titrated to determine the presence of sialic acid using the resorcinol / sulfuric acid assay (reference 12). The elution profile was plotted and fractions containing sialic acid with an average molecular weight of 4 kDa were pooled and lyophilized. The final yield of acid hydrolyzed GBM was obtained.

Example 2 This Example shows the chemical modification of GBM oligosaccharides hydrolyzed with acid. The acid-hydrolyzed GBM oligosaccharides, N-propionylated, were prepared according to the method previously described by H. Jennings et al. (reference 6) with some modifications. The GBM N-propionylated oligosaccharides were finally coupled to a MAP structure containing other oligosaccharides, in order to produce multivalent multi-carbohydrate vaccines, as described below. Reagents required: 1 - GBM oligosaccharides hydrolyzed with acid. 2 - Sodium hydroxide (2 M solution).

P1120 3 - Propionic anhydride (Aldrich). 4 - Ammonium bicarbonate (50%). 5 - Aqueous oxalic acid (50%) 6 - Sodium borohydride (Sigma) Procedure: Group B meningococcal polysaccharides hydrolyzed with acid, N-deacetylated, according to the method described by Jenning et al. , with three modifications; 1 - The reaction was carried out between approximately 110 ° and 120 ° C. 2 - The dialysis was performed using molecular porous membrane (1000 molecular weight discrimination). 3 - The neutralization of sodium hydroxide was achieved using 50% aqueous oxalic acid cold and lasted more than 1 hour. The polysaccharide (100 mg) is dissolved in 5 mL of degassed 2M sodium hydroxide containing sodium borohydride (10 mg). The resulting mixture was then heated for 6 to 8 hours at about 100 ° to 120 ° C, and the product was isolated by a combination of pH neutralization in an ice bath, using 50% oxalic acid, followed by dialysis ( four changes of 10 mM ammonium bicarbonate, 4 ° C) and lyophilization to provide one product (65.2 mg). This deacetylation P1120 caused 100% deacetylation, as determined by the complete disappearance of acetyl signal in the XH NMR spectrum. The N-deacetylated GBM oligosaccharides prepared from the previous step (55 mg) were dissolved in saturated sodium bicarbonate (12 mL) and three aliquots of propionic anhydride (0.250 mL) were added over a period of 30 minutes. The whole mixture was stirred overnight at room temperature. A ninhydrin test was performed and it was negative, which indicated the complete conversion of the free amino groups into propionamide groups. The mixture was then dialysed against distilled water (3 x 4L) and lyophilized to give the propionylated GBM polysaccharide, hydrolyzed with acid (43.2 mg).

Example 3 This example shows the preparation of oligosaccharides from Streptococcus pneumoniae. This example describes the general methods using acid hydrolysis of capsular polysaccharides (CP) of Streptococcus pneumoniae (molecular weight approximately 50 kDa) to produce oligosaccharides with a molecular mass ranging between 2.5 and 5.0 kDa. The resulting oligosaccharides can be subjected to a novel glucoconjugation technology to prepare glucoconjugates that P1120 contain multiple oligosaccharides covalently linked to a carrier or carrier protein or to a multiple antigen peptide (MAP) system. Reagents required: 1 - Serotypes CP 6B, 14, 19F and 23F (ATTC). 2 - Acetic acid. 3 - Trichloroacetic acid. 4 - Column of gel chromatography (Sephadex G-100, 10 x 1000 mm). 5 - Round bottom flask (250 mL) 6 - Magnetic stirring bar. 7 - Oil bath. Procedure: In a round bottom flask, the capsular proteins (CP) (see following Table 4) were dissolved in degassed hot water (62.5 mL) followed by the addition of the required amount of degassed acid (see following Table 4). The total mixture was degassed for a further 10 minutes and then heated using an oil bath for the required time (see following Table 4). At the end of the hydrolysis time, the total mixture was diluted five times with water and then lyophilized to produce the crude product. A gel permeation column (10 x 1000 mm, Sephadex®-G100) was calibrated with the following patterns of P1120 molecular weight: Dextran standards (P.M. 8800, 39100, 73500, 503,000), glucose (180), sucrose (342), and synthetic PRP hexamer (2340). The purification of the oligosaccharides was achieved using the Sephadex® G-100 gel column and the oligosaccharides were eluted with Mill-Q water at a flow rate of 0.9 mL / min. The fractions were collected every 3 minutes and the presence of carbohydrates was evaluated using phenol / sulfuric acid. Fractions containing oligosaccharides of 2.5 to 5 kDa molecular weight were pooled and lyophilized.

Example 4 This Example describes the preparation of oligosaccharides of N. meningi tidis. As in the case of acid hydrolysis of pneumococcal CPs, the process that was applied to N. meningi tidis involves acid hydrolysis, lyophilization and purification using gel filtration chromatography. The conditions for acid hydrolysis for the CPs of the N. meningococcal groups C, W-135 and Y were also optimized. Typically, the CPs (10 mg / mL) were mixed with 20 to 80 mM sodium acetate, pH 4.5 to 5.5, in sealed bottles under argon atmosphere between 65 ° C to 100 ° C for 8 to 12 hours. Since the CPs of group B can undergo intramolecular esterification under acidic conditions, the hydrolysis was carried out under conditions used for the hydrolysis of group C of the CPs, but the incubation time was limited to 1 hour and the pH of the reaction is immediately adjusted at pH 7 with 0.1 M NaOH, to reverse the esterification process (for more details see Example 1). The CPs of group A contain labile phosphodiester bonds, therefore, they were hydrolyzed under mild acidic conditions (such as acetic acid of 10 to 20 mM) and incubated between 50 ° C and 100 ° C for 30 to 48 hours. At the end of each hydrolysis, the reaction solutions were diluted 5 times with water and then freeze-dried. The crude oligosaccharides were fractionated by Sephadex® G-100 gel filtration chromatography (2 x 210 cm, see above). Typical chromatographic results are illustrated in Figure 4. Fractions were assessed for presence of sialic acid using a resorcinol / sulfuric acid assay (reference 12). The elution profile was plotted and the chromatographically purified oligosaccharides of 2 to 5 kDa were pooled. The sized oligosaccharides typically contained 6 to 15 repeating units. The yields were between 40 and 80%.

P1120 Example 5 This example describes the preparation of multivalent oligosaccharides conjugated in a random manner with a carrier protein. To illustrate a potential use of the present invention, the oligosaccharides of S. pneumoniae serotype 6B, 14, 19F and 23F were randomly and covalently linked with TT, as shown in Figure 1. To a TT solution (8 mg / l.2 mL of PBS), a 4 molar excess of 6B oligoscáridos oxido with periodate (0.5 mg / 0.1 mL of PBS), 14 (1.4 mg / 0.2 mL), 19F (0.65 mg / 0.12 mL) and 23F ( 1 mg / 0.2mL). The pH was adjusted to 7.4 with a few drops of 0.1 M NaOH and the reaction was stirred for 4 days at 37 ° C. On day 5, a 10-fold excess (100 μL) of NaCNBH3 (5 mg / mL) was added to the mixtures and stirred another 3 days at 37 ° C. The reaction mixture was then dialysed against excess PBS to remove unreacted oligosaccharides and NaCNBH3 for 3 days at 4 ° C. The glucoconjugate was purified by gel filtration chromatography on a Sephedex G100 column (1.6 x 100 cm). The elution profile is illustrated in Figure 5. The glycoconjugate was collected. Protein and carbohydrate analyzes were performed and the molar ratio of carbohydrate to protein was 7.1: 1. The multiple antigen conjugate (MAG) was used as an immunogen formulated with either complete adjuvant or P1120 Freund or with alum. Immunogenicity studies were performed in rabbit and mouse. The results are written below.

Example 6 This Example describes the synthesis of peptide. The peptides were synthesized (Table 1) using an ABI 43OA peptide synthesizer and an optimized chemistry t-Boc, as described by the manufacturer, they were then excised from the resin by hydrofluoric acid (HF). The peptides were purified by reversed-phase high-resolution liquid chromatography (RP-HPLC) on a Vydac C4 semiprep column (1 x 30 cm) using a gradient of acetonitrile from 15 to 55% in 0.1% trifluoroacetic acid (TFA). and it was developed at 40 minutes at a flow rate of 2 mL / min. All the synthetic peptides used in the biochemical and immunological studies were more than 95% pure as determined by the analytical HPLC. The analysis of amino acid composition carried out in a Waters Pico-Tag system were in agreement with the theoretical compositions. A synthetic MAP was prepared manually using Fmoc solid phase peptide synthesis chemistry according to a previously described modified method P1120 by Tam (reference 24). A resin Fmoc-Lys (T-Boc) -TGA (500 mg, purchased from BACHEM) with a substitution level of 180 μmol / g was normally used to prepare MAP. As a general coupling protocol, a 4-fold excess of Fmoc-protected amino acids, activated with an equal amount of HBTU and HOBT / DIEA was used for 1 hour. In order to facilitate conjugation with the oligosaccharides, at MAP substitution level it was reduced to approximately 50 μmol / g when the first Fmoc-Gly residue was coupled. When the synthesis was completed according to Figure 2, a small portion of the MAP-resin was cleaved with 95% TFA in the presence of ethanedithiol (EDT) and thioanisole, and amino acid analysis of the cleaved MAP was performed to confirm the composition of amino acids.

EXAMPLE 7 This example describes the preparation of oligosaccharides with bifunctional crosslinking groups. To a solution of oligosaccharide oxidized with periodate (3 mg / mL PBS) was added a 20 molar excess of 1,4-diaminobutane (10.5 mg). The pH of the mixture was adjusted to 7.4 and then the reaction was stirred for 4 days at 37 ° C. On day 5, an excess (500 μL of NaCNBH3 (20 mg / mL) was added to the mixture, which was stirred an additional 3 days at 37 ° C. The oligosaccharide derivatized with a functional amino group was P1120 purified by gel filtration chromatography on a Sephedex® G-50 column (1.6 x 100 cm). m-Meleimidobenzoyl-N-hydroxysuccinimide (MBS, Pierce) (20 mg, 63.6 mmol) in tetrahydrofuran (1 mL) was added to a solution of oligosaccharides derivatized with amino (4.3) mmol) in 0.1 M phosphate buffer (1 mL) , pH 7.5. The reaction mixture was stirred for 30 minutes at room temperature under argon, then extracted with ether (4 x 5 mL) to remove the MBS excess. The resulting aqueous layer was applied to a Sephadex G-25 column (20 x 300 mm) with 20 mM ammonium bicarbonate buffer, pH 7.2 and eluted with the same buffer. The elution was monitored by absorbance at 280 nm and the eluted peak was collected, pooled and lyophilized to yield the desired MBS activated oligosaccharides. The number of maleimide groups incorporated in the oligomers was determined by adding an excess of 2-mercaptoethanol to the activated oligosaccharide-MBS and making the titration of the excess using a modified Ellman method (reference 25).

Example 8 This Example describes the preparation of linear glycopeptide conjugates. A resin Fmoc-Gly-Lys (t-Boc) -TGA (500 mg) with P1120 a substitution level of 50 μmol / g was used to prepare linear peptides having a T cell-derived epitope from either S. pneumoniae or N. meningi tidis proteins, as shown in Table 1. A protocol was used. standard Fmoc chemistry coupling (see Example 6). When the synthesis was complete, a small portion of the resin with the peptide was cleaved with 95% TFA in the presence of EDT and thioanisole, to determine the quality of the synthesis. The remainder of the peptide-resin was first deprotected at the N-terminus using piperidine and then washed with dichloromethane, methanol, water and PBS. PN peptide-resin (123-140) was mixed with S. pneumoniae serotype 14 oligosaccharide oxidized with periodate (1 eq.) In methanol / phosphate buffer (pH 7.8) in the presence of NaCNBH3 at 38 ° C for 6 hours. days. After conjugation, the degree of substitution of the amino groups was determined by the ninhydrin test and the total sugar content was assessed using the orcinol test. The linear glycopeptide-resin was cleaved with 95% TFA in the presence of EDT and thioanisole. After working, the glycopeptide was titrated to determine amino acid composition and carbohydrate content.

P1120 Example 9 This Example describes the preparation of multivalent conjugates of glycopeptide-MAP. A MAP resin containing three different T cell epitopes [PN (123-140), PN (6-281) and SP37, Table 1] derived from membrane proteins of S. pneumoniae was synthesized using a Fmoc-Glyco resin. Lys-TGA with a substitution level of 50 mmol / g as shown in Figure 2. The complete synthesis was carried out manually using an optimized Fmoc chemistry coupling protocol, as described above (Example 6). When the synthesis was complete, a small portion of MAP-resin was cleaved with 95% TFA in the presence of EDT and thioanisole. The cleaved MAP presented the correct amino acid composition as a result of amino acid analysis. The MAP resin was reduced with DTT to remove the t-butylthio protecting groups from the cysteine residues. After excessive washing, the MAP resin was resuspended in a DMF / PBS solution, then a 4-fold excess of the sulfo-SIAB activated oligosaccharides was admixed from S. pneumoniae type 14 (0sl4). After mixing overnight at room temperature, the MAP resin was collected by filtration and washed with PBS, DMF and then methanol. The resin Map-0sl4 was subjected to P1120 Ellman test and determination of sulfhydryl group. The substitution level of SH was half the initial value. Reattachment did not increase the amount of 0s14 conjugated to the MAP resin. The presence of N-acetylgalactosamine (GlcNAc) in the glyco-MAP resin, a carbohydrate found in 0sl4, was independently confirmed by carbohydrate analysis. MAP-0sl4 was first treated with 1% TFA to remove Mtt (a lysine protecting group) from the Mtt-lysine residues, then neutralized with a mild base, 1% diisopropylethylamine (DIEA) / DMF. The presence of amino groups was assessed by the ninhydrin test which indicated that > 90% of the Mtt groups had withdrawn. The MAP-Osl4 resin was resuspended in PBS and then mixed with 4 equivalents of S. pneumoniae serotype 6B (0s6B) oligosaccharides oxidized with periodate in DMF / phosphate buffer (pH 7.8) in the presence of NaCNBH3 at 38 ° C for 6 hours. days . After conjugation, the substitution of the amino groups was determined by the ninhydrin test and was from 80 to 90% of the original value. Once again, double or triple coupling did not improve the conjugation of Os6B with MAP-Osl4 resin. Although the coupling efficiency was poor (approximately 15%), the presence of ribitol in the MAP conjugate, a carbohydrate found P1120 in Os6B, was confirmed by carbohydrate analysis. The MAP-Osl4-Os6B conjugate was treated with 20% piperidine in DMF to remove the Fmoc protecting group from the Fmoc-lysine residues. After washing, MAP-Osl4-Os-6B resin was mixed with a 4-fold excess of the oligosaccharides of S. pneumoniae serotype 19F, oxidized with periodate (Osl9F) in DMF / phosphate buffer (pH 7.8) in the presence of NaCNBH3 at 38 ° C for 6 days. After conjugation, the degree of substitution of the amino group measured by the ninhydrin test was approximately 90%. The coupling reaction was repeated and its effectiveness was determined to be about 15%. However, the presence of N-acetylmanose (ManNAc), a sugar found in Osl9F, was detected by carbohydrate analysis. A small portion of MAP-resin glycoconjugate was cleaved with 95% TFA in the presence of EDT and thioanisole. After working, the MAP-glycoconjugate was evaluated to determine the amino acid composition and carbohydrate content, and it was found to have a correct amino acid composition and a correct carbohydrate content. Although the overall yield was very low (approximately 5%), however, these results show that they can be conjugated selectively and P1120 sequentially different oligosaccharides with a MAP resin.

Example 10 This example describes the preparation of the native polysaccharide-polylysine conjugate. 0.5 mL of oxidized polysaccharides with periodate (25 mg in 1 mL of 0.1 M sodium phosphate buffer, pH 6. 0), was prepared from native polysaccharides of S. pneumoniae or N. meningi tidis treated with aqueous sodium periodate, added to polylysine (5 mg) in 2 mL of 0.2 M sodium phosphate buffer, pH 8.0, followed by the addition of sodium cyanoborohydride (10 equivalents of polylysine). After incubation at 37 ° C for 5 days, the reaction mixture was dialyzed against 0.1 M phosphate buffer, (4 x 1 L), pH 7.5 and the resulting solution was applied to an analytical column of Superóse 12 (15 x 300 mm, Pharmacia) equilibrated with phosphate buffer 0.2 M sodium, pH 7.2, and eluted with the same buffer. The fractions were monitored to determine absorbance at 230 nm. The main peak was collected. The amount of protein was determined using the Bio Rad protein assay. The presence of polysaccharides was confirmed by the orcinol test.

P1120 Example 11 This example describes mouse immunogenicity studies of multivalent oligosaccharide-TT conjugates. Five BALB / c mice were immunized intramuscularly (im) with multivalent oligosaccharide conjugates TT (20 μg of oligosaccharides) emulsified in complete Freund's adjuvant (FCA) and followed by two booster doses (half the amount of the same immunogen in adjuvant complete Freund) at 2-week intervals. The antisera were collected, inactivated at 56 ° C and then stored at -20 ° C, the results are shown in Figure 8.

EXAMPLE 12 This example describes rabbit immunogenicity studies of conjugates of multivalent oligosaccharides-TT formulated in alum. Rabbits were immunized intramuscularly with 0.5 mL of multivalent oligosaccharide-TT conjugates (20 μg of oligosaccharide equivalent) mixed with 3 mg A1P04 per mL, followed by two booster doses (half the amount of the same immunogen) at intervals of 2 weeks. The antisera were collected every 2 weeks, after the first injection, they were inactivated with heat at 56 ° C for 30 minutes and stored at -20 ° C.

Example 13 This example describes rabbit immunogenicity studies of multivalent-carrier oligosaccharide conjugates formulated in FCA. Rabbits were immunized intramuscularly with 0.5 mL of conjugates of multivalent oligosaccharides-TT or oligosaccharides-MAP (conjugates having 12 μg of oligosaccharide equivalents mixed with 1 mL of FCA) followed by two booster doses (half of the amount thereof) immunogen formulated with Freund's complete adjuvant (IFA)) at 2 week intervals. The antisera were collected every 2 weeks after the first injection, thermally inactivated at 56 ° C for 30 minutes and stored at -20 ° C.

Example 14 This example describes specific ELISA assays for peptides. The cavities of a microtiter plate (Nunc-Immunoplate, Nunc, Denmark) were coated with 500 ng of individual peptides in 50 μL of coating buffer (15 mM Na2C03, 35 mM NaHCO3, pH 9.6) for 16 hours at room temperature. The plates were blocked with 0.1% BSA (w / v) in phosphate buffer.

P1120 (PBS) for 30 minutes at room temperature. The antisera were serially diluted and added to the cavities and incubated for 1 hour at room temperature. After removal of the antisera, the plates were washed five times with PBS containing 0.1% (w / v) Tween-20 and 0.1% (w / v) BSA. F (ab ') 2 from goat anti-rabbit IgG antibodies was conjugated with horseradish peroxidase (Jackson ImmunoResearch Labs Inc., PA), diluted (1/8,000) with wash buffer and added to the microtiter plates. After 1 hour at room temperature, the plates were washed five times with the washing solution. The plates were then developed using tetramethylbenzidine (TMB) in H202 (ADI, Toronto) as a substrate. The reaction was stopped with H2SO2 IN and the optical density was measured at 450 nm using a Titretek Multiskan II (Flow Labs., Virginia). Two irrelevant pertussis toxin peptides, NAD-Sl (19 residues) and S3 (123-154) (32 residues), were included as negative controls for peptide-specific ELISA assays. The titrations were performed in triplicate and the reactive titration of an antiserum was defined as the dilution that consistently showed an increase of twice the absorbance value with respect to that obtained with the pre-immune serum.

P1120 Example 15 This Example describes the measurement of anti-polysaccharide antibodies. The cavities of a microtiter plate (Nunc-Immunoplate, Nunc, Denmark) were coated with 200 ng of polysaccharide-polylysine conjugates of S. pneumoniae or N. meningi tidis in 200 μL of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) for 16 hours at room temperature. The plates were blocked with 0.1% BSA (w / v) in phosphate buffer.

(PBS) for 30 minutes at room temperature. Antisera diluted in series against the PRP-carrier conjugates were obtained and added to the amounts and incubated for 1 hour at room temperature. After removal of the antisera, the plates were washed five times with PBS containing 0.1% (w / v) Tween-20 and 0.1% (w / v) BSA. F (ab ') 2 d Goat anti-rabbit or anti-mouse IgG antibodies, conjugated with horseradish peroxidase (Jackson ImmunoResearch Labs Inc., PA) (1 / 8,000) were diluted with wash buffer and added to the microtitre plates. After 1 hour of incubation at room temperature, the plates were washed five times with the washing buffer. Subsequently the plates were developed using tetramethylbenzidine (TMB) in H202 (ADI, Toronto), the reaction was stopped with H2S02 IN and the P1120 optical density was measured at 450 nm using Titretek Multiskan II (Flow Labs., Virginia). The triplicate titrations were performed and the reactive titre of an antiserum was defined as the dilution that consistently shows a two-fold increase in the value of the optical density with respect to that obtained with the pre-immune serum.

Example 16 This Example describes a proliferation assay for synthetic T cell epitopes. The T cell epitope was mapped by priming BALB / c mice with 5 μg of individual carrier proteins. Three weeks later, the spleens were removed and the splenocytes cultured in RPMI 1640 medium (Flow Lab) supplemented with 10% thermal inactivated fetal calf serum (Gibco), 2 mM L-glutamine (Flow Lab), penicillin 100 U / mL (Flow Lab), streptomycin 100 μg / mL (Flow Lab), rIL-2 10 units / mL and 50 μM 2-mercaptoethanol (sigma) for 5 to 7 days. The proliferative responses of the splenocytes primed against the panel of peptides were determined in a standard of an in vitro assay (reference 26). Briefly, 106 splenocytes were co-cultivated in a 96-well microtiter plate, where 5 x 10 5 syngeneic spleen cells, P11 0 fresh, irradiated (1700 Rad) were used as a source of antigen presenting cells (APC) in the presence of increasing molar concentrations (0.03 to 3 μM of peptide solvent in the culture medium without IL-2). The cultures were maintained for 40 hours in a humidified incubator with C02 5% / air while maintaining a temperature of 37 ° C. During the final 16 hours of culture, 0.5 μCi of [3H] -Tdr (5 Ci / mmol, NEN) was added to each well. The cells were then harvested on glass fiber filters and the incorporation of 3 H-thymidine into cell DNA was measured in a scintillation counter β (Beckman). The results are expressed as the mean of the triplicate determination made at each concentration of the peptide. The standard deviation was always less than 15%. The proliferative responses were considered as positive when the incorporation of 3H-thymidine was three times greater than that obtained with any irrelevant peptide or with the culture medium.

EXHIBITION SUMMARY As a summary of this disclosure, the invention provides novel immunogenic, multivalent oligosaccharides, as well as novel conjugation methods for their preparation, their use with vaccines and their use to provide antibodies for use in diagnostics.

P1120 Modifications are possible within the scope of this invention. P1120 TABLE 1 T-POTENTIAL CELL EPITOPES FROM MENINGOCOCAL PROTEINS AND • or H oo TABLE 2. Responses of anti-peptide antibody in rabbits immunized with combined conjugates of linear glycopeptide [6B-PN (123-140) + 14-PN (263-281) + 23F-P (123-140)] Anti-Peptide Antibody Title Pre-Immune Peptide Title Geometric Media PN (123-140) < 100 12,800 PN (263-281) < 100 3,200 a Total responses of anti-peptide antibodies were determined by peptide-specific ELISA assays. b Antisera were taken from immunized rabbits.

TABLE 3. Responses of anti-peptide antibody in rabbits immunized with MAP glycopeptide conjugates.

Anti-peptide antibody titre5 Peptide titre1 Pre-Immune Geometric Media PN (123-140) < 100 633,400 PN (263-281) < 100 12,800 SP37 < 100 51,200 a Total anti-peptide antibody responses were determined by peptide-specific ELISA assays. b Antisera were obtained from rabbits immunized three times with the MAP glycopeptide conjugate.

P1120 TABLE 4 P1120 REFERENCES MMWR, (1994) 43: 23-26 MM R (1989) 38: 64-76 Austrian R, (1981; Rev. Zpfect, Dis. 3. {Suppl.): Sl- S17 Dagan ec al., ( 1992.} J.-Med. Assoc. 268: 3328-3332) H. Jennings et al., (1986), J. Ipunun. 1, 1011 H. Jennings et al. (J. Iironuno., 1986, 137, 1708 Peeters er al., (1991) Infecv. Immun. 55: 3504-3510 Paradiso et al., (1993.). Vaccine? Is. 4: 239-248 Schneerson ßt al. (1980) J. expt. Mea, 152: 3S1, Barington et al. 1993, J? Fect * I mun, 61: 432-438, Svennemolm, 1957, xoch & m. Biophys. Acra. 604: 24. Walker ßt al. . { 1987), Ipfecc. lamun 58: 1184-1189, Yother and Briles (1992), J. Bacterio] .. 174: 601- € 09, Sampson et al., Infect. Immuñ. 62: 319-324, Rokbx et al. (1995) FEMS Microbiol. ßtc. 132: 277-283. Stimson in al., (1995), Mol. Microbiol. 17: 1201-1214 McGuinness et al., (1990;, J. Exp. Med. 171: 1871- 1882. Wiertz et al., In Rivier, J and Marshall, GR (Ed.) Pepzides: Ch & Pisury, Structure and Biology, (Proceedings- of the llth American Pept of Symposium.}, ESCOM, Leiden, 1990, p.371-372, De Velasco ec al., Infecz. Iimaun, 63: 961-968 0 McQueen et al., (1991) pediatr. Ras. 31 (part 2): Abs er 1056? Eby et ai., (1994) In Vaccine A'.Modem Approaches to Vaccines pp. 119-124. Edited by E. Norry, F. Bra n, R.M. C anocfc and Ginsberg, H.S. Cold Spr ng Harbor, N.Y. Cold Spring Harbor Press Kandil et al., (1997) Glycoconjugate J. 14: 13-17 Tam (1996, J. Isvm? N. Mevh. 196: 1732) Ridlea et al., (1983) M &vhods Enzymol. 91: 49-60). Sia et al, Sean. J. Imraunol. 26: 683-69Q

Claims (20)

1. CLAIMS: 1. A multivalent immunogenic molecule characterized by: a carrier molecule containing at least one functional T cell epitope, and multiple different carbohydrate fragments, each linked to the carrier molecule and each containing at least one epitope functional B cell, wherein the carrier molecule imparts enhanced immunogenicity to the multiple carbohydrate fragments.
2. 2. The molecule according to claim 1, characterized in that the carbohydrate fragments are fragments of bacterial capsular oligosaccharide.
3. 3. The molecule according to claim 2, characterized in that the capsular oligosaccharide fragments are capsular oligosaccharide fragments of Streptocococcus pneumoniae.
4. 4. The molecule according to claim 3, characterized in that the capsular oligosaccharide fragments are derived from at least two capsular polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F.
5. 5. The molecule according to claim 3 or 4, characterized in that the carrier molecule is a protein containing T cell epitope or protein S fragment. P1120 pneumoniae.
6. 6. The molecule according to claim 2, characterized in that the capsular polysaccharide fragments are fragments of capsular oligosaccharide of Neisseria meningi tidis. The molecule according to claim 6, characterized in that the fragments of oligosaccharides are derived from at least two capsular polysaccharides of N. meningi tidis Groups A, G, C, W-15 and Y. 8. The molecule according to claim 6 or 7, characterized in that the carrier molecule is a protein containing T-cell epitope or protein fragment of N. meningitidis. 9. The molecule according to any of claims 2 to 8, characterized in that the fragments of oligosaccharides are sized from 2 to 5 kDa. 10. The molecule according to claim 1, characterized in that the carrier molecule is an oligopeptide containing at least one functional T cell epitope or a carrier protein, for example tetanus toxoid. 11. The molecule according to claim 1, characterized in that the carbohydrate fragments are fragments of tumor antigens based on carbohydrates. 12. The molecule according to claim 11, P1120 characterized in that the tumor antigen is Globo H, Le or STn 13. The molecule according to any of claims 1 to 12, produced by site-directed glycoconjugation. A method for forming a multivalent immunogenic molecule, characterized by: treating at least two different carbohydrate molecules to obtain carbohydrate fragments thereof and conjugating each of the carbohydrate fragments with a carrier molecule. The method according to claim 14, characterized in that the carbohydrate molecules are capsular polysaccharides of a bacterium and the oligosaccharide fragments of capsular polysaccharide are selected with dimensions of between 2 to 5 kDa. The method according to claim 15, characterized in that the conjugation step is performed by random conjugation of the oligosaccharide fragments with the carrier molecule. 17. The method according to claim 15, characterized in that the conjugation step is slid by site-directed glycoconjugation. 18. The method according to claim 17, characterized in that the site-directed glycoconjugation is P1120 performs first by forming a multiple antigen peptide as the carrier molecule anchored to a polymeric anchor, wherein at least two carrier peptide segments have different terminal protecting groups; selectively removing one of the protecting groups; coupling a first of the oligosaccharide fragments with the unprotected carrier peptide segment; selectively removing another of the protecting groups; coupling a second fragment of the oligosaccharide fragments with the unprotected carrier peptide segment; and cleaving the resulting molecule, separating it from the polymer anchor. 19. An immunogenic composition for protection against meningitis, characterized by: (1) a multiple pneumococcal glucoconjugate according to any of claims 3, 4 or 5, (2) a multiple meningococcal glucoconjugate according to claims 6, 7 or 8 and (3) an immunogenic synthetic conjugate of PRP-peptide. The immunogenic composition according to claim 19, characterized by at least one additional antigen. 21. A method for determining the presence of antibodies specifically reactive with a multivalent immunogenic molecule, according to any of the P1120 claims 1 to 13, characterized port () to contact the sample with 3a equivalent immunogenic molecule to produce complexes that make up the moldaula and any of the preslentßß antl puerpos < sn the sample, which copesífloamantß react with the molecule; and (b) determine the production of the complexes. 22. The method according to claim 21, wherein the multi-immunogenic molecule is a multiplied glucocopneumococcal multiplex according to claims 3, 4 or 5, or a multiple-conjugated mendngococcal conjugate according to claims 6, 7. 8. 8. A diagnostic kit for determining the presence of antibodies to the immunogenic molecule according to any of claims 1 to 13, in a sample, which is characterized by (a) the multivalent ittimonogenic molecule? (b) means for contacting the multivalent molecule with the sample, in order to produce complexes comprising the multivalent molecule and any of the antibodies present in the sample; and (c) means to determine the production of complexes. 24. The kit according to claim 23, characterized in that the immunogenic conjugate molecule of F1120 any of claims 1 to 15 is present in the form of an immunogenic composition of claims 19 or 20. 25. A diagnostic kit for determining the presence of a multivalent immunogenic molecule in a sample, comprising the points: (a) antibodies specific for the carbohydrate fragments of a multivalent immunogenic molecule according to any of claims 1 to 15. (b) means for contacting the antibodies with the sample, in order to produce complexes comprising the multivalent immunogenic molecule and the antibodies; and (c) means for determining the production of the complex. 26. The kit according to claim 25, characterized in that the antibodies are antibodies to the components of the immunogenic composition of claims 19 or 20. P1120