MXPA01009459A - Vaccine - Google Patents

Abstract

The present invention relates to the field of bacterial polysaccharide antigen vaccines. In particular, the present invention relates to vaccines comprising a pneumococcal polysaccharide antigen, typically a pneumococcal polysaccharide conjugate antigen, formulated with a protein antigen form Streptococcus pneumoniae, and optionally a Th1-inducing adjuvant.

Description

VACCINE Field of the Invention The present invention relates to bacterial polysaccharide antigen vaccines, their preparation and the use of such polysaccharides in medicines. In particular, the present invention relates to three inter-related aspects: A - vaccines comprising a pneumococal polysaccharide antigen, typically a pneumococal polysaccharide conjugated antigen, formulated with a protein antigen from Streptococcus pneumoniae and optionally a Th 1 -inducer adjuvant; B - advantageous, specific pneumococcal polysaccharide conjugates adjuvanted with a Th 1 adjuvant; and C - conjugated bacterial polysaccharides conjugated in general with protein D from H. influenzae.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is a Gram positive bacterium responsible for considerable morbidity and mortality (particularly in youth and old age), causing invasive diseases such as pneumonia, bacteremia and meningitis and diseases associated with colonization, such as Otitis media acute The proportion of pneumococcal pneumonia in the US for people over 60 years of age is estimated at 3 to 8 per 100,000. In 20% of cases, it leads to bacteremia and other manifestations, such as meningitis, with a mortality rate close to 30%, even with antibiotic treatment. The pneumococcus is encapsulated with a chemically bound polysaccharide, which confers serotype specificity. There are 90 known serotypes of pneumococci and the capsule is the main determinant virulence for pneumococci since the capsule not only protects the internal surface of the complement bacterium, but is itself poorly immunogenic. Polysaccharides are T-independent antigens and can not be processed or presented in MHC molecules to interact with T cells. However, they can stimulate the immune system through an alternating mechanism that involves the degradation of surface receptors in B cells. It was shown in several experiments that the protection against invasive pneumococcal disease correlates more strongly with specific antibodies to the capsule and the protection is serotype specific. Vaccines based on polysaccharide antigen are well known in the art. Four that have been authorized for human use include the Vi polysaccharide of Salmonella typhi, the PRP polysaccharide from Haemophilus influenzae, the meningococcal tetravalent vaccine composed of serotypes A, C, W135 and Y and the 23-valent pneumocoal vaccine composed of polysaccharides. corresponding to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 1 1 A, 12 F, 14, 15 B, 17 F, 18 C, 19 A, 19 F, 20, 22 F, 23 F, and 33 (considering at least 90% of the pneumococcal blood isolates).

The last three vaccines provide protection against bacteria that cause respiratory infections that result in severe morbidity and mortality in infants, these vaccines have not yet been authorized to be used in children under two years of age because they are inadequately immunogenic in this group of age [Peltola et al. (1984), N. Engl. J. Med. 310: 1 561-1 566]. Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Similarly, the elderly amount to poor responses to pneumococcal vaccines [Roghmann et al. , (1987), J. Gerontol. 42: 265-270], despite the increasing incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62: 271-285]. Strategies that have been designed to overcome this lack of immunogenicity in infants include the binding of the polysaccharide to large immunogenic proteins, which provides a surrounding T cellular support and which induces an immunological memory against the polysaccharide antigen with which it is conjugated. Conjugated pneumococcal glycoprotein vaccines are currently being evaluated for immunogenicity and efficacy in the various age groups. A) Pneumococcal Polysaccharide Vaccines The 23-valent pneumococal unconjugated vaccine has shown wide variation in clinical efficacy, from 0% to 81% (Fedson et al. (1 994) Arch. Intern Med. 154: 2531 -2535). The efficacy seems to be related to the risk group that is being immunized, such as advanced age, Hodgkin's disease, splenectomy, sickle cell disease and agammaglobulinemic disease (Fine et al. (1994) Arch. Intern Med. 1 54 : 2666-2677) and also with the manifestation of the disease. The 23-valent vaccine does not demonstrate protection against diseases of pneumococcal pneumonia (in certain high-risk groups, such as the elderly) and otitis media. Accordingly, there is a need for improved pneumococcal vaccine compositions, particularly one that is more effective in preventing or decreasing pneumococcal disease (particularly pneumonia) in the elderly and in young children. The present invention provides such improved vaccine. B) Selected Pneumococal Polysaccharide Conjugate + 3D-MPL Compositions It is generally accepted that the protective efficacy of the unconjugated pneumococal vaccine that is marketed is more or less related to the antibody concentration induced after vaccination; however, the authorization of the 23 polysaccharides was accepted only after the immunogenicity of each component polysaccharide (Ed Williams et al New York Academy of Sciences 1995 pp. 241 -249). Therefore, further improvement of antibody responses to pneumococcal polysaccharides may increase the percentage of children and older adults who respond with protective levels of antibodies to the first injection of polysaccharide or polysaccharide conjugate and could reduce the dose and number of Injections required to induce protective immunity to infections caused by Streptococcus pneumoniae.

Since the early twentieth century, researchers have experimented with an enormous number of compounds that can be added to antigens to improve their immunogenicity in vaccine compositions [reviewed in M. F. Powell & M.J. Newman, Plenum Press, NY; "Vaccine Design - the Subunit and Adjuvant Approach" (1995) Chapter 7"A Compendium of Vaccine Adjuvants and Excipients"]. Many are very efficient but cause adverse, local and systemic, significant reactions that avoid their use in human vaccine compositions. Adjuvants based on aluminum (such as alum, aluminum hydroxide or aluminum phosphate), first described in 1926, remain the only immunological adjuvants used in human vaccines licensed in the United States. Aluminum-based adjuvants are examples of the kind of adjuvant vehicle that works through the "deposition effect" they induce. The antigen is adsorbed on its surface and, when the confection is injected, the adjuvant and the antigen do not dissipate immediately into the blood stream - rather, the composition persists in the local environment of the injection and results in a more pronounced immune response. Such vehicle adjuvants have the additional known advantage of being suitable for stabilizing antigens that are prone to rupture, for example, some polysaccharide antigens. 3D-MPL is an example of a non-vehicle adjuvant. Its full name is lipid A of deacylated 3-O-monophosphoryl (or de-O-acylated monophosphoryl lipid A or monophosphoryl lipid A of 3-O-desacyl-4 ') and is referred to as 3D-MPL to indicate that the position 3 of the reducing end glucosamine is de-O-acylated. For preparation, see GB 222021 1 A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Originally developed in the early 90's, when the method for 3-O-deacylate 4'-monophosphoryl lipid A derivative (MPL) led to a molecule with a more attenuated toxicity, without change in immunostimulatory activity. 3D-MPL has been used as an adjuvant either alone or, preferably, in combination with a reservoir-type vehicle adjuvant, such as aluminum hydroxide, aluminum phosphate or oil-in-water emulsions. In such compositions, the antigen and 3D-MPL are contained in the same particulate structures, allowing a more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further improve the immunogenicity of an antigen adsorbed on [Thoelen et al. Vaccine (1 998) 16: 708-14; EP 689454-B1]. Such combinations are also preferred in the art for antigens that are prone to adsorption (e.g., bacterial polysaccharide conjugates), where alum adsorption tends to stabilize the antigen. The adjuvants based on precipitated luminium are the most used, since they are the only adjuvants currently used in authorized human vaccines. According to the above, vaccines containing 3D-MPL in combination with aluminum-based adjuvants are favored in the field due to their ease of development and speed of introduction into the market. MPL (non-deacylated) has been evaluated as an adjuvant with several vaccine antigens conjugated with monovalent polysaccharide. Co-injection of MPL in saline improved the serum antibody response for four monovalent polysaccharide conjugates: tetanus toxoid 6B of PS pneumococal, diphtheria toxoid 12 of PS pneumococal and S.aureus type 5 and S.aureus type 8 conjugated with exotin A of Pseudomonas aeruginosa [Schneerson et al. , J. Immunology (1991) 147: 2136-2140]. The improved responses were considered antigen-specific. The MPL in an oil-in-water emulsion (a vehicle-type adjuvant) consistently improved the effect of MPL on the saline solution due to the presence of MPL and antigen in the same particulate structure and it was considered as the adjuvant system to choose for the optimal supply of other conjugated polysaccharide vaccines. Devi et al. [Infect. Immun. (1 991) 59: 3700-7] evaluated the adjuvant effect of MPL (not 3-deacylated) in saline on the murine antibody response to a TT conjugate of capsular polysaccharide of Cryptococcus neoformans. When the MPL was used concurrently with the conjugate, there was only a marginal increase in both the specific IgM response and IgG specific to the PS; however, MPL had a much greater effect when administered 2 days after the conjugate. The practicality of using an immunization scheme that requires a delay in the administration of MPL in relation to the antigen, especially in infants, is questionable. The adjuvant effect of MPL with polysaccharides and polysaccharide-protein conjugates seems to depend on the composition. Again, the incorporation of MPL into a suitable slow-release delivery system (e.g., using a vehicle adjuvant) provides a more durable adjuvant effect and overcomes the problem of delayed timing and administration. In summary, the state of the art has considered that, for a polysaccharide or antigens conjugated with polysaccharides in particular, when MPL or 3D-MPL is used as an adjuvant, it is advantageously used in conjunction with a vehicle adjuvant (eg, adjuvants based on aluminum) in order to maximize its immunostimulatory effect. Surprisingly, the present inventors have found that for certain pneumococcal polysaccharide conjugates, the immunogenicity of the vaccine composition is significantly higher when the antigen is formulated with 3D-MPL alone rather than with 3D-MPL in conjunction with a vehicle adjuvant (such as an adjuvant based on aluminum). In addition, the observed improvement depends on the concentration of 3D-MPL used and on whether the conjugates in particular are in a monovalent composition or on whether they combine to form a polyvalent composition. C) Bacterial polysaccharide - Protein D conjugates As mentioned above, vaccines based on polysaccharide antigen are well known in the art. The authorized polysaccharide vaccines, mentioned above, have different demonstrated clinical efficacy. The Vi polysaccharide vaccine has been estimated to have an efficacy between 55% and 77% in the prevention of culture-confirmed tyroid fever (Plotkin and Cam, (1995) Arch Intern Med 155: 2293-99). Meningococcal C polysaccharide vaccine has been shown to have an efficacy of 79% under epidermal conditions (De Wals P, ef al. (1996) Bull World Health Organ. 74: 407-41 1). The 23-valent pneumococal vaccine has shown a wide variation in clinical efficacy, from 0% to 81% (Fedson et al. (1994) Arch Intern Med. 154: 2531-2535). As mentioned above, it is accepted that the protective efficacy of the pneumococcal vaccine is more or less related to the concentration of antibodies induced after vaccination. Among the problems associated with the polysaccharide approach to vaccination is the fact that polysaccharides per se are poor immunogens. Strategies that have been designed to overcome this lack of immunogenicity include the binding of the polysaccharide to large highly immunogenic protein carriers, which provide surrounding T cell support. Examples of these highly immunogenic vehicles that are currently commonly used for the production of polysaccharide immunogens include Diphtheria toxoid (DT or mutant CRM197), Tetanus toxoid (TT), Bocallave Limpet hemocyanin (KLH) and the derivative of purified Tuberculin protein (PPD). Problems Associated with Commonly Used Vehicles Several problems are associated with each of these commonly used vehicles, including the production of GMP conjugates and also the immunological characteristics of the conjugates. Despite the common use of these vehicles and their success in inducing anti-polysaccharide antibody responses, these are associated with several disadvantages. For example, it is known that antigen-specific immune responses can be suppressed (epitope deletion) by the presence of pre-existing antibodies directed against the vehicle, in this case, Tetanus toxin (Di John et al. (1989) Lancet , 2: 1415-8). In the population as a whole, a very high percentage of people will have pre-existing immunity for both DT and TT, since people are normally vaccinated with these antigens. In the United Kingdom, for example, 95% of children receive the DTP vaccine that includes both DT and TT. Other authors have described the problem of epitope deletion for peptide vaccines in animal models (Sad et al., Immunology, 1991; 74: 223-227; Schutze et al. , J. Immunol. 1 35: 4, 1885; 231 9-2322). In addition, for vaccines that require regular reinforcement, the use of highly immunogenic carriers such as TT and DT is likely to suppress the polysaccharide antibody response after several injections. These multiple vaccinations may also be accompanied by undesirable reactions, such as delayed-type response (DTH) hypercapacity. KLH is known as a potent immunogen and has already been used as a vehicle for IgG peptides in human clinical trials. However, some adverse reactions (reactions similar to DTH or IgE sensitization) as well as antibody responses have been observed. against antibody. Therefore, the selection of a carrier protein for a vaccine based on polysaccharide will require a balance between the need to use a vehicle that works in all patients (broad recognition of MHC), the induction of high levels of antibody responses of anti-polysaccharides and low antibody response against the vehicle. Accordingly, vehicles previously used for polysaccharide-based vaccines have many disadvantages. This is particularly true in combination vaccines, where the suppression of the epitope is especially problematic if the same vehicle is used for various polysaccharide antigens. In WO 98/51 339, multiple vehicles were used in combination vaccines in order to try to obtain this effect. The present invention provides a novel vehicle for use in the preparation of immunogenic conjugates based on polysaccharide / polypeptide, which does not suffer from the aforementioned disadvantages. The present invention provides a protein D (EP 0 594 61 0 B 1) from Haemophilus influenzae, or fragment thereof, as a carrier for immunogenic compositions based on polysaccharide, including vaccines. The use of this vehicle is particularly advantageous in combination vaccines.

BRIEF DESCRIPTION OF THE INVENTION A) Pneumococcal Polysaride Vaccines In accordance with the above, the present invention provides a vaccine composition, comprising at least one polysaride antigen Streptococcus pneumoniae (preferably conjugated) and a protein antigen Streptococcus pneumoniae or immunologically functional equivalent of them, optionally with a Th 1 adjuvant (an adjuvant that induces an immune response of Th 1). Preferably, both a pneumococal protein and a Th1 adjuvant are included. The compositions of the invention are particularly suitable in the treatment of elderly pneumonia. Pneumococcal polysaride vaccines (conjugated or not) may not be able to protect against pneumonia in the elderly population for whom the incidence of this disease is very high. The key defense mechanism against pneumococcus is opsonophagocytosis (an event mediated by humoral / neutrophil B-cell, originated by the production of antibodies against the pneumococal polysaride, eventually phagocytizing the bacteria), however, parts of the opsonic mechanisms involved are unbalanced in advanced age, that is, the production of superoxide by PMN (polymorphonuclear cells), another production of reactive oxygen species, the mobilization of PMN, PMN apoptosis, deformability of PMN. Antibody responses may also become unbalanced in later life. Contrary to the commonly accepted dogma, normal levels of anti-capsule polysaride antibodies may not be effective in the complete evacuation of bacteria, since pneumococci can invade host cells to evade this branching of the immune system. Surprisingly, the present inventors have found that, by simultaneously stimulating the cell-mediated branching of the immune system (e.g., T cell-mediated immunity), in addition to the humoral ramification of the immune system (mediated by B cell), a synergy results ( or cooperation) that is capable of improving the evacuation of pneumococci from the host. This is a discovery that will help the prevention (or treatment) of pneumococcal infection in general, but it will be particularly important in the prevention (or treatment) of pneumonia in old age where vaccines based on polysarides do not show efficacy. The present inventors have found that both weapons of the immune system can be synergized in this manner if a pneumococcal polysaride (preferably conjugated) is administered with a pneumococal protein (preferably a protein expressed on the surface of the pneumococci, or secreted or released, which can processed and presented in the context of Class II and MHC class I on the surface of infected mammalian cells). Although a pneumococal protein can activate cell-mediated immunity on its own, the inventors have also found that the presence of a Th 1 -inducer adjuvant in the vaccine formulation aids this weapon of the immune system and, surprisingly, further enhances the synergy between both weapons of the immune system. B) Selected Pneumococal Polysaride Conjugate + Compositions of 3D-MPL Accordingly, the present invention also provides an antigenic composition comprising one or more pneumococcal polysaride conjugates, adjuvanted with 3D-MPL and substantially free of aluminum-based adjuvants, wherein at least one of the Conjugated pneumococcal polysarides is significantly more immunogenic in compositions comprising 3D-MPL compared to compositions comprising 3D-MPL in conjunction with an aluminum-based adjuvant. Preferred embodiments provided are antigenic compositions comprising conjugates of one or more of the following pneumococcal capsular polysarides: serotype 4, 6B, 18C, 1 9F and 23F. In such compositions, each of the polysaccharides is surprisingly more immunogenic in compositions comprising 3D-MPL alone compared to compositions comprising 3D-MPL and an aluminum-based adjuvant. Therefore, in one embodiment of the invention there is provided an antigenic composition comprising the capsular polysaccharide serotype of Streptococccus pneumoniae 4, 6B, 1 8C, 1 9F or 23F, conjugated to an immunogenic protein and 3D-MPL adjuvant, wherein the composition is substantially free of adjuvants based on aluminum. In a second embodiment, the present invention provides an antigenic combination composition, substantially free of aluminum-based adjuvants and comprising 3D-MPL adjuvant and two or more pneumococcal polysaccharide conjugates selected from the group consisting of: serotype 4; serotype 6B; serotype 18C; serotype 19F; and serotype 23F. C) Bacterial polysaccharide - protein D conjugates Accordingly, the present invention provides a polysaccharide conjugated antigen comprising a polysaccharide antigen derived from a pathogenic bacterium conjugated to protein D from Haemophilus influenzae or a protein D fragment thereof. . In addition, the invention provides polyvalent vaccine compositions, wherein one or more of the polysaccharide antigens is conjugated with protein D.

DESCRIPTION OF THE INVENTION A) Pneumococcal polysaccharide vaccines The present invention provides an improved vaccine, particularly for the prevention or decrease of pneumococcal infection of advanced age (e / o infants and toddlers). In the context of the invention, a patient is considered to be of advanced age if he is 55 years of age or older, typically over 60 years and more generally over 65 years of age. Therefore, in one embodiment of the invention there is provided a vaccine composition, suitable for use in advanced age (and / or infants and toddlers), comprising at least one polysaccharide antigen of Streptococcus pneumoniae and at least a protein antigen of Streptococcus pneumoniae. In a second preferred embodiment, the present invention provides a vaccine (suitable for the prevention of pneumonia in advanced age) comprising at least one polysaccharide antigen of Streptococcus pneumoniae and at least one protein antigen of Streptococcus pneumoniae and a Th1 adjuvant. Such a vaccine is also expected to be useful in the treatment of pneumococcal infection (eg, otitis media) in other high-risk groups of the population, such as for infants or toddlers. In a third embodiment, a vaccine composition comprising a pneumococal polysaccharide antigen and a Th 1 adjuvant is provided. Antigens Streptococcus pneumoniae Polysaccharides of the Invention Typically, the Streptococcus pneumoniae vaccine of the present invention will comprise polysaccharide antigens (preferably conjugates), wherein the polysaccharides are derived from at least four pneumococcal serotypes. Preferably, the four serotypes include 6B, 14, 1 9F and 23F. More preferably, at least 7 serotypes are included in the composition, for example, those derived from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. More preferably still, at least 1 1 serotypes are included in the composition, for example, the composition in one embodiment includes capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated). In a preferred embodiment of the invention, at least 1 3 polysaccharide antigens (preferably conjugates) are included, although additional polysaccharide antigens, for example 23-valent (such as serotypes 1, 2, 3, 4, 5) are also contemplated by the invention. , 6B, 7F, 8, 9N, 9V, 1 0A, 1 1A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F). For vaccination in advanced age (for example, for the prevention of pneumonia), it is advantageous to include serotypes 8 and 1 2F (and more preferably, 1 5 and 22 also) to the antigenic composition 1 1 -valent described above in order to form a 15-valent vaccine, while for infants or toddlers (where otitis media is of greatest concern), serotypes 6A and 1A are advantageously included to form a 3-valent vaccine. For the prevention / reduction of pneumonia in the elderly population (+55 years) and otitis media in Infants (up to 1 8 months) and children who start walking (typically 1 8 months up to 5 years), a preferred modality of The invention is to combine a multivalent polysaccharide of Streptococcus pneumoniae, as described herein, with a protein of Streptococcus pneumoniae or immunologically functional equivalent thereof. Pneumococcal protein of the invention For the purposes of this invention, "immunologically functional equivalent" is defined as a protein peptide comprising at least one protective epitope derived from the proteins of the invention. Such epitopes are characteristically exposed on the surface, highly conserved and can produce a bactericidal antibody response in a host or prevent toxic effects. Preferably, the functional equivalent has at least 15 and preferably 30 or more contiguous amino acids from the protein of the invention. More preferably, fragments, omissions of the protein, such as variants of transmembrane omission thereof (ie, the use of the extracellular domain of proteins), fusions, chemically or genetically detoxified derivatives and the like, can be used with the condition of that are capable of substantially promoting the same immune responses as the native protein. Preferred proteins of the invention are those pneumococcal proteins that are exposed on the outer surface of the pneumococcus (capable of being recognized by the immune system of a host for at least part of the pneumococcus life cycle) or are proteins that are secreted or released by the pneumococcus. More preferably, the protein is a toxin, adhesin, 2-component signal transducer or Streptococcus pneumoniae lipoprotein or immunologically functional equivalents thereof. Particularly preferred proteins for inclusion in such a combination vaccine include, but are not limited to: pneumolysin (preferably detoxified by treatment or chemical mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jui 1 1; 1 8 (13): 401 0"Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2", Mitchell et al. Biochim Biophys Acta 1 989 Jan 23; 1 007 (1): 67-72"Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties", WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton ef al.), WO 99 / 03884 (NAVA)]; PspA and transmembrane omission variants thereof (EU 5804193 - Briles ef al.); PspC and transmembrane omission variants thereof (WO 97/09994 - Briles et al.); PsaA and transmembrane omission variants thereof (Berry &Paton, Infect Immun 1 996 Dec; 64 (12): 5255-62"Sequence heterogeneity of PsaA, at 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae"); pneumococal choline binding protein and variants of transmembrane omission thereof; CbpA and transmembrane omission variants thereof (WO 97/41 151; WO 99/51266); Glyceraldehyde-3-phosphate dehydrogenase (Infect.Immun.199664: 3544); HSP70 (WO 96/40928); PcpA (Sánchez-Beato ef al. FEMS Microbiol Lett 1 998, 164: 207-14); M-like protein, SB Patent Application No. EP 08371 30; and adhesin 1 8627, SB Patent Application No. EP 0834568. The proteins used in the present invention are preferably selected from the group of pneumolysin, PsaA, PspA, PspC, CbpA or a combination of two or more such proteins. The present invention also encompasses immunologically functional equivalents of such proteins (as defined above). Within the composition, the protein can help induce a T cell-mediated response against pneumococcal disease - particularly required for protection against pneumonia - which cooperates with the humoral ramification of the immune system to inhibit invasion by pneumococci and to stimulate opsonophagocytosis.

The additional advantages of including the protein antigen is the presentation of additional antigens for the opsonophagocytosis process and the inhibition of bacterial adhesion (if an adhesin is used) or the neutralization of toxin (if a toxin is used). Accordingly, in one embodiment of the invention, there is provided a Streptococcus pneumoniae vaccine comprising a pneumococcal polysaccharide conjugate vaccine comprising polysaccharide antigens derived from at least four serotypes, preferably at least seven serotypes, more preferably at least eleven serotypes and at least one, but preferably two, Streptococcus pneumoniae proteins. Preferably one of the proteins is Pneumolysin or PsaA or PspA or CbpA (more preferably, detoxified pneumolysin). A preferred combination contains at least pneumolysin or a derivative thereof and PspA. As mentioned above, a problem associated with the polysaccharide approach to vaccination is the fact that polysaccharides per se are poor immunogens. To overcome this, the polysaccharides can be conjugated with protein vehicles, which provide assistance from surrounding T cells. Accordingly, it is preferred that the polysaccharides used in the invention bind to such a protein carrier. Examples of such carriers that are currently commonly used for the production of polysaccharide immunogens include Diphtheria and Tetanus toxoids (DT, DT CRM197 and TT, respectively), Bocallave Limpet Hemocyanin (KLH), OMPC from N. meningitidis and the purified tuberculin protein derivative (PPD). However, several problems are associated with each of these commonly used vehicles (see section "Problems Associated with Commonly Used Vehicles" above). The present invention provides in a preferred embodiment a new vehicle for use in the preparation of immunogen constructions based on polysaccharide, which does not suffer from these disadvantages. The preferred vehicle for immunogenic compositions based on pneumococal polysaccharide (or vaccines) is protein D from Haemophilus influenzae (EP 594610-B), or fragments thereof. Fragments suitable for use include fragments that span T-helper epitopes. In particular, a protein D fragment will preferably contain 1/3 N-terminal protein. A further preferred vehicle for the pneumococal polysaccharide is the pneumococal protein itself (as defined above in the "Pneumococcal Proteins of the Invention" section). The vaccines of the present invention are preferably adjuvanted. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel (alum) or aluminum phosphate, but it can also be a calcium, iron or zinc salt, or it can be a non-soluble suspension of acylated tyrosine or acylated sugars, cationic or anionically derived polysaccharides or polyphosphazenes. It is preferred that the selected adjuvant be a preferential inducer of a Th 1 type of response to aid cell-mediated branching of the immune response.

TH1 Adjuvants of the Invention Elevated levels of Th1-type cytokines tend to favor the induction of cell-mediated immune responses to a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen. antigen. It is important to remember that the distinction of the immune response of type Th 1 and Th2 is not absolute. In reality, an individual will withstand an immune response that is described as predominantly Th 1 or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of what is described in murine CD4 + ve murine clones of Mosmann and Coffman (Mosmann, TR and Coffman, RL (1989) TH 1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties Annual Review of Immunology, 7, p. 145-173). Traditionally, Th 1 -type responses are associated with the production of INF-? Cytokines. and IL-2 by T lymphocytes. Other cytokines frequently directly associated with the induction of Th 1-type immune responses are not produced by T cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10. Suitable adjuvant systems that promote a predominantly Th 1 response include monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A and a combination of monophosphoryl lipid A, preferably monophosphoryl lipid A 3 -des-O-acilado (3D-MPL), together with an aluminum salt. An improved system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as set forth in WO 94/001 53, or a less reactogenic composition where the QS21 is repressed with cholesterol as is disclosed in WO 96/33739. An aprticularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210 and is a preferred formulation. Preferably, the vaccine additionally comprises a saponin, more preferably QS21. The formulation may also comprise an oil in water emulsion and tocopherol (WO 95/1 721 0). The present invention also provides a method for the production of a vaccine formulation comprising mixing a protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL. Non-methylated CpG containing oligonucleotides (WO 96/02555) is also a preferential inducer of a TH 1 response and is suitable for use in the present invention. Particularly preferred compositions of the invention comprise one or more conjugated pneumococcal polysaccharides, one or more pneumococcal proteins and a Th 1 adjuvant. Induction of a cell-mediated response by means of a pneumococal protein (as described above) and cooperation between both arms of the immune system can be aided by the use of such a Th-1 adjuvant, resulting in a particularly effective vaccine against pneumococcal disease in general and, importantly, against pneumococcal pneumonia in old age. In a further aspect of the present invention, an immunogen or vaccine, as described herein, is provided for use in medicine. In a still further aspect of the invention, there is provided a composition comprising a pneumococal polysaccharide conjugate and a Th1 adjuvant (preferably 3D-MPL) which is capable of seroconverting or inducing a humoral antibody response against the polysaccharide antigen within a population of non-responders. It is known that 10-30% of the population does not respond to polysaccharide immunization (it does not respond to more than 50% serotypes in a vaccine) (Konradsen et al., Scand. J. Immun 40: 251 (1 994); Rodríguez ef al. , JID, 173: 1 347 (1996)). This may also be the case for conjugate vaccines (Musher et al., Clin.Inf. Dis. 27: 1487 (1998)). This can be particularly serious for high-risk areas of the population (infants, children who start walking and elderly). The present inventors have found that a combination of a conjugated pneumococal polysaccharide (which is prone to a low response in a particular population) with a Th 1 adjuvant (see "Th 1 Adjuvants of the invention" above) can surprisingly overcome this inability to respond. Preferably, 3D-MPL and, more preferably, 3D-MPL free of aluminum-based adjuvant (which still provides a Vaccine Preparations of the Invention). The vaccine preparations of the present invention can be used to protect or treat a mammal susceptible of infection, by means of the administration of said vaccine through the systemic or mucosal route, these administrations may include injection through the intramuscular, intraperitoneal, intradermal or subcutaneous routes, or through the oral mucosal administration in the oral tracts. / food, respiratory, genitourinary, intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (since the nasopharyngeal transport of pneumococci can be prevented more effectively, thus attenuating the infection in its early stages). amount of conjugated antigen in each dose of vaccine is selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending on the specific immunogen that is used and the way in which it is presented. In general, it is expected that each dose comprises 0.1-100 μg of polysaccharide, preferably 0.1-50 μg, preferably 0.1-10 μg, of which the most preferable range is 1 to 5 μg. The content of protein antigens in the vaccine will typically be in the range of 1 -100 μg, preferably 5-50 μg, more typically in the range of 5-25 μg. The optimal amounts of components for a particular vaccine can be determined by standard studies that involve the observation of appropriate immune responses in the subjects. After an initial vaccination, subjects may receive one or several adequately separate booster immunizations. The vaccine preparation is generally described in the Vaccine Design ("The Subunit and Adjuvant Approach") (eds Powell M.F. &Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, U.S. Patent. Do not. 4,235,877. B) Selected Pneumococal Polysaccharide Conjugate + 3D-MPL Compositions For the purposes of this invention, the term "pneumococcal polysaccharide conjugates of the invention" describes those conjugates of capsular polysaccharides of Streptococcus pneumoniae that are more immunogenic in compositions comprising 3D-MPL compared to compositions comprising 3D-MPL in conjunction with an aluminum-based adjuvant (eg, conjugates of serotype 4, serotype 6B, serotype 18C, serotype 19F, or serotype 23F). For the purposes of this invention, the term "substantially free of aluminum-based adjuvants" describes a composition that does not contain sufficient adjuvant based on aluminum (e.g., aluminum hydroxide and, particularly, aluminum phosphate) to cause any decrease in the immunogenicity of a pneumococal polysaccharide conjugate of the invention, as compared to an equivalent composition comprising 3D-MPL without adjuvant based on added aluminum. Preferably, the antigenic composition should contain adjuvant consisting essentially of 3D-MPL. The amounts of adjuvant based on aluminum added per dose should preferably be less than 50 μg, more preferably less than 30 μg, still more preferably less than 10 μg and more preferably there is no aluminum-based adjuvant added to the antigenic compositions of the invention. For the purposes of this invention, the determination of whether a pneumococal conjugated polysaccharide is significantly more immunogenic in compositions comprising 3D-MPL compared to compositions comprising 3D-MPL in conjunction with an aluminum-based adjuvant, should be established as described in Example 2. an indication of whether a composition is significantly more immunogenic when comprising 3D-MPL alone, the ratio of GMC IgG concentration (as determined in Example 2) between compositions comprising 3D-MPL alone versus an equivalent composition comprising 3D -MPL in conjunction with adjuvant based on aluminum, should be more than 2, preferably more than 5, more preferably more than 7, even more preferably more than 9 and more preferably more than 14. Among the problems associated with the polysaccharide approach for vaccination, one finds the fact that polysaccharides per se are poor immunogens. Strategies that have been designed to overcome this lack of immunogenicity include the binding (conjugation) of the polysaccharide to large protein vehicles, which provides assistance from surrounding T cells. It is preferred that the pneumococcal polysaccharides of the invention bind to a protein carrier that provides surrounding T cell support. Examples of such vehicles that may be used include Diphtheria toxoids, Diphtheria and Tetanus mutants (DT, CRM197 and TT, respectively), Bocallave Limpet Hemocyanin (KLH), the purified Tuberculin protein derivative (PPD) and OMPC of Neisseria meningitidis. More preferably, protein D from Haemophilus influenzae (EP 0 594 610-B) or fragments thereof (see section C), is used as the immunogenic protein carrier for the pneumococcal polysaccharides of the invention. In one embodiment, the antigenic composition of the invention comprises pneumococal polysaccharide serotype 4 (PS) conjugated to an immunogenic protein and formulated with 3D-MPL adjuvant, wherein the composition is substantially free of aluminum-based adjuvant. In additional embodiments, the anthogenic composition comprises PS 6B, 18C, 19F, or 23F, respectively, conjugated to an immunogenic protein and formulated with a 3D-MPL adjuvant, where the composition is substantially free of aluminum-based adjuvant. In a still further embodiment of the invention, there is provided an antigenic combination composition comprising two or more pneumococcal polysaccharide conjugates from the group of PS 4, PS 6B, PS 1 8C, PS 19F and PS 23F, formulated with 3D-adjuvant. MPL, where the composition is substantially free of aluminum-based adjuvant. The immunogenicity of the pneumococcal polysaccharide conjugates of the invention is not significantly affected by the combination with other pneumococcal polysaccharide conjugates (Example 3). Accordingly, a preferred aspect of the invention provides an antigenic combination composition comprising one or more pneumococcal polysaccharide conjugates of the invention in combination with one or more additional pneumococcal polysaccharide conjugates, wherein the composition is formulated with 3D-adjuvant. MPL, but is substantially free of aluminum-based adjuvant. In the further preferred embodiments of the invention, combination antigenic compositions are provided which contain at least one and preferably 2, 3, 4 or 5 PS 4, 6B, 18C, 19F or 23F conjugated pneumococcal polysaccharides and, in addition, any combination of other pneumococcal polysaccharide conjugates, which are formulated with 3D-MPL adjuvant but are substantially free of aluminum-based adjuvant. Typically, the antigenic combination composition of Streptococcus pneumoniae of the present invention will comprise conjugated polysaccharide antigens, wherein the polysaccharides are derived from at least four, seven, eleven, thirteen, fifteen or twenty three serotypes (see, "Streptococcus pneumoniae Polysaccharide Antigens of the Invention" above for the preferred combinations of serotypes, depending on the disease to be treated). The antigenic compositions of the invention are preferably used as vaccine compositions to prevent (or treat) pneumococcal infections, particularly in the elderly and infants and children who begin to walk. Additional embodiments of the present invention include: the proportion of the above antigenic compositions for use in medicine; a method for inducing an immune response to a capsular polysaccharide conjugate of Streptococcus pneumoniae, comprising the steps of administering a safe and effective amount of one of the antigenic compositions to a patient; and the use of one of the above antigenic compositions in the manufacture of a medicament for the prevention (or treatment) of pneumococcal disease. For the prevention / reduction of pneumonia in the elderly population (+55 years) and otitis media in infants (up to 18 months) and children who start walking (typically 18 months to 5 years), an additional preferred modality of The invention is to combine a multivalent Streptococcus pneumoniae polysaccharide conjugate formulated as described herein with a Streptococcus pneumoniae protein or immunologically functional equivalent thereof. See the previous section "Pneumococcal proteins of the invention" for the preferred protein / protein combinations. Preferably, the antigenic compositions (and vaccines) described hereinabove are lyophilized until they are almost used, at which point they are reconstituted extemporaneously with diluent. More preferably, they are lyophilized in the presence of 3D-MPL and reconstituted extemporaneously with saline.

The lyophilization of the compositions results in a more stable composition (for example, prevents the breakdown of the polysaccharide antigens). The process is also surprisingly responsible for a higher antibody titer against pneumococcal polysaccharides. This has proven to be particularly significant for PS 6B conjugates. Another aspect of the invention is, therefore, a lyophilized antigenic composition comprising a conjugate of PS 6B adjuvanted with 3D-MPL and substantially free of aluminum-based adjuvants. For the preparation of vaccines, see the section above "Invention vaccine preparations"). C) Conjugates of Bacterial Polysaccharide - Protein D The tendency towards combination vaccines has the advantage of reducing discomfort in the recipient, facilitating programming and ensuring the end of the regimen; but there is still the concomitant risk of reducing vaccine efficacy (see above for discussion on epitope suppression through vehicle protein overuse). Accordingly, it would be advantageous to prepare vaccine combinations that meet the needs of a population and that, moreover, do not exhibit immunogenic interference between their components. These advantages can be achieved by the immunogenic compositions (or vaccines) of the invention, which are of particular benefit for the administration of combination vaccines to high-risk groups, such as infants, toddlers or elderly persons. advanced The present invention provides a protein D from Haemophilus influenzae or fragments thereof as a vehicle for an immunogenic composition based on polysaccharides, including vaccines. Fragments suitable for use include fragments that encompass T helper epitopes. In particular, the D protein fragment will preferably contain 1/3 of the N-terminus of the protein. Protein D is an IgD-binding protein from Haemophius influenzae (EP 0 594 610 B1) and is a potential immunogen. The polysaccharides to be conjugated with Protein D, contemplated by the present invention, include, but are not limited to, Vi polysaccharide antigen against Salmonella typhi, meningococcal polysaccharides (including type A, C, W1 35 and Y, and the modified polysaccharide and polysaccharides of meningococcus of group B), polysaccharides from Staphylococcus aureus, polysaccharides from Streptococcus agalactae, polysaccharides from Streptococcus pneumoniae, polysaccharides from Mycobacteria, for example, Mycobacterium tuberculosis (such as trehalose mannofosfoinisitidas, mycolic acid, arabinomannanos covered with mannose, the capsule of the same and arabinogalactans), polysaccharide from Cryptococcus neoformans, the lipopolysaccharides of non-typeable Haemophilus influenzae, the capsular polysaccharide from Haemophilus influenzae b, the lipopolysaccharides from Moraxella catharralis, the lipopolysaccharides from Shigella sonnei, the lipopeptidofosfoglicano (LPPG) of Trypanosoma cruzi, the gangliosides associated with cancer GD3, GD2, the tumor associated mucins, especially the T-F antigen and the sialyl T-F antigen and the HIV-associated polysaccharide that is structurally related to the T-F antigen. The polysaccharide can be linked to the carrier protein by any known method (for example, by Likhite, U.S. Patent No. 4,372,945 and by Armor et al., U.S. Patent No. 4,474,757).

Preferably, the conjugation of CDAP is carried out (WO 95/08348). In the CDAP, the cyanoylation reagent, 1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP), is preferably used for the synthesis of polysaccharide-protein conjugates. The cyanolation reaction can be carried out under relatively mild conditions, which prevents the hydrolysis of the sensitive alkaline polysaccharides. This synthesis allows direct coupling to a carrier protein. The polysaccharide is solubilized in water or a saline solution. The CDAP is dissolved in acetonitrile and added immediately to the polysaccharide solution. The CDAP reacts with the hydroxyl groups of the polysaccharide to form a cyanate ester. After the activation stage, the vehicle protein is added. The amino groups of lysine react with the activated polysaccharide to form a covalent bond of isourea. After the coupling reaction, a large excess of glycine is then added to repress the residual activated functions. The product is then passed through a gel permeation to remove unreacted vehicle protein and residual reagents. According to the above, the invention provides a method for the production of protein D conjugates, polysaccharides, which comprises the steps of activating the polysaccharide and linking the polysaccharide to protein D.

In a preferred embodiment of the invention, an immunogenic composition (or vaccine) is provided for the prevention of Streptococcus pneumoniae infections. The mechanisms by which pneumococci are diffused in the lung, cerebrospinal fluid and blood are poorly understood. The growth of bacteria that reach normal lung alveoli is inhibited by their relative drought and by the phagocytic activity of the alveolar macrophages. Any anatomical or physiological change of these coordinated defenses tends to increase the susceptibility of the lungs to infection. The cell wall of Streptococcus pneumoniae has an important role in the generation of an inflammatory response in the alveoli of the lung (Gillespie et al (1997), I &65: 3936). Typically, the Streptococcus pneumoniae vaccine of the present invention will comprise protein D polysaccharide conjugates, wherein the polysaccharide is derived from at least four, seven, eleven, thirteen, fifteen or 23 serotypes. See above "Streptococcus pneumoniae Polysaccharide Antigens of the Invention" for the preferred combinations of serotypes, dependent on the disease to be treated. In a further embodiment of the invention, a Neisseria meningitidis vaccine is provided; in particular, serotypes A, B, C, W-1 35 and Y. Neisseria meningitidis is one of the most important causes of bacterial meningitis. The carbohydrate capsule of these organisms can act as a virulence determinant and as an objective for protective antibodies. However, it is known that carbohydrates are immunogenic scars in children. The present invention provides a particularly suitable protein carrier for these polysaccharides, protein D, which provides T cell epitopes that can activate a T cell response to aid the proliferation and maturation of polysaccharide antigen-specific B cells, as well as to the induction of an immunological memory. In an alternative embodiment of the invention, a capsular polysaccharide of Haemophilus influenzae b (PRP) -D protein conjugate is provided. The present invention also contemplates combination vaccines that provide protection against a range of different pathogens. A protein D carrier is surprisingly useful as a vehicle in combination vaccines where multiple polysaccharide antigens are conjugated. As mentioned above, deletion of epitopes is likely to occur if the same vehicle is used for each polysaccharide. WO 98/51 339 presented compositions to try to minimize this interference by conjugating a proportion of the polysaccharides in the composition with DT and the rest with TT. Surprisingly, the present inventors have found that protein D is particularly suitable for minimizing such epitope suppression effects in combination vaccines. One or more polysaccharides in a combination can be advantageously conjugated with protein D and, preferably, all antigens are conjugated with protein D within such combination vaccines. A preferred combination includes a vaccine that provides protection against infection of Neisseria meningitidis C and Y (and preferably A), wherein the polysaccharide antigen of one or more serotypes Y and C (and more preferably A) bind to protein D. The vaccine based on Haemophilus influenzae polysaccharide (PRP preferably conjugated with TT, DT or CRM197, or more preferably with protein D) can be formulated with the above combination vaccines. Many pediatric vaccines are now given as a combination vaccine in order to reduce the number of injections a child has to receive. Therefore, for pediatric vaccines, other antigens can be formulated with the vaccines of the invention. For example, vaccines of the invention can be formulated with, or administered separately but at the same time, the well-known 'trivalent' combination vaccine comprising Diphtheria toxoid (DT), tetanus toxoid (TT) and components of pertussis [typically, detoxified pertussis toxoid (PT) and filamentous haemagglutinin (FHA) with optional pertactin (PRN) and / or agglutinin 1 +2], for example, the commercial vaccine INFANRIX- DTPa ™ (SmithKIineBeecham Biologicals) containing antigens of DT, TT, PT, FHA and PRN, or with a complete cellular pertussis component, for example, as marketed by SmithKIineBeecham Biologicals SA , like Tritanrix ™. The combined vaccine may also comprise another antigen, such as Hepatitis B surface antigen (HBsAg), Polio virus antigens (for example, inactivated trivalent poliovirus - IPV), outer membrane proteins of Moraxella catarrhalis, non-typeable proteins of Haemophilus influenzae, outer membrane proteins of N.meningitidis B. Examples of preferred protein antigens of Moraxella catarrhalis that can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP106 [WO 97/41731 (Antex ) and WO 96/34960 (PMC)]; OMP21; LbpA and LbpB [WO 98/55606 (PMC)]; TbpA and TbpB [WO 97/13785 and WO 97/32980 (PMC)]; CopB [Helminen ME ef al. (1993) Infect. Immun. 61: 2003-2010]; UspA1 / 2 [WO 93/03761 (University of Texas)]; and OmpCD. Examples of non-typeable antigens of Haemophilus influenzae that can be included in a combination vaccine (especially for the prevention of otitis media) include: Fimbrin protein [(EU 5766608 - Ohio State Research Foundation)] and fusions comprising peptides thereof [e.g., peptide fusions of LB 1 (f); EU 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01 638 (Cortees)]; P6 [EP 281673 (State University of New York)]; TbpA and TbpB; Hia; Hmw1, 2; Hap; and D1 5. The preferred pediatric vaccines contemplated by the present invention are: a) Conjugate of polysaccharide of N. meningitidis C and polysaccharide conjugate of Haemophilus influenzae b, optionally with N.meningitidis A and / or conjugate of polysaccharide Y, taking into account that at least one polysaccharide antigen and preferably all are conjugated with protein D. b) Vaccine of a) with components of DT, TT, pertussis (preferable PT, FHA and PRN), Hepatitis B surface antigen and IPV (inactivated trivalent poliovirus vaccine). c) Polysaccharide antigens of Streptococcus pneumoniae conjugated with protein D. d) Vaccine of c) with one or more antigens of Moraxella catarrhalis and / or non-typeable Haemophilus influenzae. All of the above combination vaccines can benefit from the inclusion of protein D as a vehicle. Clearly, the more vehicles that are involved in a combination vaccine (for example, to overcome epitope suppression), the more expensive and complex the final vaccine is. Having all or most of the polysaccharide antigens of a combination vaccine with protein D thus provides a considerable advantage. For the prevention of pneumonia in the elderly population (+55 years) and otitis media in infants or toddlers, a preferred embodiment of the invention is to combine multivalent polysaccharide antigens from Streptococcus pneumonia - protein D, as described herein, with a protein of Streptococcus pneumoniae or immunologically functional equivalent thereto. See the section above "Pneumococcal Proteins of the Invention" for the preferred protein / protein combinations that may be included in such a combination. Accordingly, the present invention provides an immunogenic composition comprising a polysaccharide conjugate of Streptococcus pneumoniae-protein D and a protein antigen of Streptococcus pneumoniae.

The conjugated polysaccharide-protein D antigens of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel (alum) or aluminum phosphate, but may also be a calcium, iron or zinc salt, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationic or anionically derived polysaccharides or polyphosphazenes. For older vaccines, it is preferred that the selected adjuvant be a preferential inducer of a TH1 response type. For particular Th1 adjuvants see above "Th 1 Adjuvants of the Invention". In a further aspect of the present invention, a vaccine or immunogen, as described herein, is provided for use in medicine. For the preparation / administration of the conjugate vaccine, see "Preparation of the Vaccine of the Invention" above. Protein D is also advantageously used in a vaccine against otitis media, since it is itself an immunogen capable of producing B-cell-mediated protection against nontypable H. influenzae (ntHi). The ntHi can invade the host cells and evade the B cell-mediated effects induced by the protein antigen. The present inventors have surprisingly discovered a way to increase the efficiency of protein D (either on its own or as a vehicle for a polysaccharide) as an antigen for an otitis media vaccine. This is done by the adjuvance of protein D in such a way that a strong Th1 response is induced in the subject, such that the cell-mediated weapon of the immune system is optimized against protein D. This is surprisingly achieved by the use of a lyophilized composition comprising protein D and a Th1 adjuvant (preferably 3D-MPL), which is reconstituted shortly before administration. The invention thus also provides such compositions, a process for making such compositions (by lyophilization of a mixture comprising protein D and a Th 1 adjuvant) and a use of such a composition in the treatment of otitis media. In a broader sense, the inventors foresee that lyophilization of an immunogen in the presence of a Th 1 adjuvant (see "Th 1 Adjuvants of the Invention"), preferably 3D-MPL, will generally increase the immune response against the immunogen. Accordingly, the present invention is applicable to any immunogen for which a stronger Th 1 immune response is required. Such immunogens comprise bacterial, viral and tumorous protein antigens, as well as auto proteins and peptides. EXAMPLES The examples illustrate but do not limit the invention. EXAMPLE 1 Capsular Polysaccharide of S. pneumoniae: Candidate 1 -valent vaccine includes the capsular polysaccharide serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 1 8C, 1 9F and 23F which were elaborated essentially as described in EP 7251 3. Each polysaccharide is activated and derived by the use of CDAP chemistry (WO 95/08348) and conjugated with the protein carrier. All polysaccharides are conjugated in their native form, except for serotype 3 (which was reduced in size to reduce its viscosity). Protein Vehicle: The selected protein carrier is recombinase D (PD) protein from non-typeable Haemophilus influenzae, expressed in E. coli. EXPRESSION OF PROTEIN D Protein D of Haemophilus influenzae Raw material Protein D that encodes DNA Protein D is highly conserved among H. influenzae of all serotypes and non-typeable strains. The vector pHIC348 containing the DNA sequence encoding the entire protein D gene has been obtained from Dr. A. Forsgren, Department of Medical Microbiology, Lund University, Malmo General Hospital, Malmo, Sweden. The DNA sequence of protein D has been published by Janson et al. (1 991) Infect. Immun. 59: 1 1 9-125. The Expression Vector pMG1 The expression vector pMG1 is a derivative of pBR322 (Gross et al., 1988) in which the control elements derived from? bacteriophage for the transcription and translation of previously inserted genes (Shatzman et al., 1 983). In addition, the Ampicillin resistance gene was exchanged with the Canamycin resistance gene. The E.coli strain AR58 The E.coli strain AR58 was generated by the transduction of N99 with a phage deposit P1 previously developed in a derivative of SA500 (galE:: TN10, lambdaKil "cl587? H1.) N99 and SA500 are K12 strains of E. coli derived from the laboratory of Dr. Martin Rosenberg at the National Institute of Health The Expression Vector pMG1 For the production of protein D, the DNA encoding the protein has been cloned into the expression vector pMG1. This plasmid uses signals from lambdafago DNA to direct the transcription and translation of inserted foreign genes.The vector contains the PL promoter, the OL operator and two sites of use (NutL and NutR) to release the transcriptional polarity effects when provided Protein N (Gross et al., 1989) The vectors containing the PL promoter are introduced into a lysogenic host of E. coli to stabilize the plasmid DNA. in lambdaphago DNA defective in reproduction, integrated into the genome (Shatzman et al. , 1983). The chromosomal lambdaphago DNA directs the synthesis of the cl repressor protein that binds to the OL repressor of the vector and prevents the binding of the RNA polymerase to the PL promoter and also the transcription of the inserted gene. The cl gene of the AR58 expression strain contains a temperature-sensitive mutant so that PL-directed transcription can be regulated by temperature shift, i.e., an increase in the temperature of the culture inactive to the repressor and start the synthesis of the foreign protein. This expression system allows the controlled synthesis of foreign proteins, especially those that can be toxic to the cell (Shimataka &; Rosenberg, 1981). The E.coli strain AR58 The lysogenic E.coli strain AR58 used for the production of the protein D vehicle is a N99 derivative of standard NIH E.coli strain K12 (F "su" galK2, lacZ'thr " It contains a defective lysogenic lambdafago (galE :: TN10, lambdaKM "cl857? H1). The Kil phenotype prevents the interruption of the macromolecular synthesis of the host.The mutation of cl857 gives a temperature-sensitive lesion to the repressor of cl.The omission of? H 1 removes the right operon from the lambdafago and hosts bio, uvr3 and sites ch 1 A. Strain AR58 was generated by transduction of N99 with a P1 phage deposit previously developed in a derivative of SA500 (galE:: TN 10, lambdaKil "cl857? H 1). The introduction of the defective lysogen in N99 was selected with tetracycline by virtue of the presence of a TN 1 0 transposon encoding the tetracycline resistance in the adjacent galE gene. Construction of vector pMGMDPPrD The vector pMG 1 containing the gene encoding the protein of S1 non-structural Influenzae virus (pMGNSI) was used to construct pMGMDPPrD. The protein D gene was amplified by PCR from the vector pHIC348 (Janson et al., 1991) with PCR start loads containing Ncol and Xbal restriction sites at the 5 'and 3' ends, respectively. The Ncol / Xbal fragment was then introduced into pMGNSI between Ncol and Xbal, thus creating a fusion protein containing the 81 N-terminal amino acids of the NS1 protein followed by the PD protein. This vector was labeled pMGNSI PrD. Based on the construction described above, the final construct was generated for the expression of protein D. A BamHI / BamHI fragment was removed from pMGNSI PrD. This DNA hydrolysis removes the coding region of NS1, except for the first three N-terminal residues. After re-ligating the vector, a gene encoding a fusion protein with the following N-terminal amino acid sequence has been generated: MDP SSHSSNMANT NS 1 Protein D Protein D does not contain a guiding peptide or the N-terminal cysteine to which the lipid chains normally bind. Accordingly, the protein is neither evacuated in the periplasm nor lipidated and remains in the cytoplasm in a soluble form. The final construction of pMG-MDPPrD was introduced into host strain AR58 by thermal shock at 37 ° C. The plasmid containing the bacteria was selected in the presence of Canamycin. The presence of protein D encoding the DNA graft was demonstrated by digestion of isolated plasmid DNA with selected endonucleases. The recombinant E. coli strain is referred to as ECD4. The expression of protein D is under the control of the promoter PL lambda / Operator O. Host strain AR58 contains a cl gene sensitive to temperature in the genome that blocks the expression of PL lambda at low temperature by binding to O. Once the temperature rises, cl is released from OL and protein D is expressed. At the end of the fermentation, the cells are concentrated and freeze. The extraction of the harvested cells and the purification of protein D was carried out as follows. The frozen cell culture pellet is thawed and resuspended in a cell disruption solution (Citrate Regulator pH 6.0) to a final OD6so = 60. The suspension is passed twice through a high pressure homogenizer to P = 1000 bars. The homogenate of the cell culture is clarified by centrifugation and the cell debris is removed by filtration. In the first purification step, the filtered lysate is applied to a cation exchange chromatography column (Rapid Flow Sepharose SP). The PD binds to the gel matrix by ionic interaction and is levigated by a step increase in the ionic resistance of the levigation regulator. In a second purification step, the impurities are retained in an anion exchange matrix (Q Sepharose Fast Flow). The PD does not bind in the gel and can be collected in the flow. In both steps of column chromatography, the collection of the fraction is monitored by OD. The flow through tf anion exchange column chromatography containing purified protein D is concentrated by ultrafiltration. Protein D containing the retentate from the ultrafiltration is finally passed through a 0.2 μm membrane. Chemistry: Activation and coupling chemistry: Activation and coupling conditions are specific for each polysaccharide. These are given in Table 1. The native polysaccharide (except for PS3) was dissolved in 2M NaCl or in water for injection. The optimal concentration of polysaccharide was evaluated for all serotypes. From a deposit solution of 100 mg / ml in acetonitrile, CDAP (CDAP / PS ratio of 0.75 mg / mg PS) was added to the polysaccharide solution. 1.5 minutes later, 0.2M triethylamine was added to obtain the specific activation pH. Activation of the polysaccharide was carried out at this pH for 2 minutes at 25 ° C. Protein D (the amount depends on the initial PS / PD ratio) was added to the activated polysaccharide and the coupling reaction was carried out at the specific pH for 1 hour. The reaction was then quenched with glycine for 30 minutes at 25 ° C and overnight at 4 ° C. The conjugates were purified by gel filtration, using a gel filtration column of Sephacryl 500HR, equilibrated with 0.2M NaCl. The carbohydrate and protein content of the levigated fractions was determined. The conjugates were dited and sterile filtered on a 0.22 μm sterilization membrane. The proportions of PS / Protein in the conjugate preparations were determined. Characterization: Each conjugate was characterized and met the specifications described in Table 2. The polysaccharide content (μg / ml) was measured by the Resorcinol test and the protein content (μg / ml) by the Lowry test. The final proportion of PS / PD (w / w) was determined by the proportion of the concentrations. Residual content of DMAP (ng / μg PS): The activation of the polysaccharide with CDAP introduces a cyanate group in the polysaccharide and DMAP (4-dimethylamino-pyridine) is released. The residual content of DMAP is determined by a specific test developed in SB. Polysaccharide-free content (%): The polysaccharide-free content of the conjugates was maintained at 4 ° C or stored 7 days at 37 ° C was determined in the supernatant obtained after incubation with a-PD antibodies and was saturated in sulphate of ammonium, followed by centrifugation. An a-PS / a-PS ELISA was used for the quantification of free polysaccharide in the supernatant. The absence of conjugate was also controlled by an a-PD / a-PS ELISA. The reduction of the amount of free polysaccharide results in an improved conjugate vaccine. Antigenicity: The antigenicity in the same conjugates was analyzed in a sandwich-type ELISA, where the capture and detection of antibodies was a-PS and a-PD, respectively. Free Protein Content (%): The "free" residual D protein level was determined by using a method with SDS treatment of the sample. The conjugate was heated 10 minutes at 100 ° C in the presence of 0.1% SDS and injected onto a gel filtration column of SEC-HPLC (TSK 3000-PWXL). Since protein D is a dimer, there is a risk of overestimating the level of "free" D protein by dissociating the structure with SDS. Molecular size (Kav): Molecular size was taken to cabi in a gel filtration column of SEC-HPLC (TSK 5000-PWXL). Stability: Stability was measured in a filtration by HPLC-SEC gel (TSK 6000-PWXL) for conjugates maintained at 4 ° C and stored for 7 days at 37 ° C. The 1 1 -valent characterization is given in Table 2. The protein conjugates can be adsorbed onto aluminum phosphate and deposited to form the final vaccine. Conclusion: Immunogenic conjugates have been produced that have been shown to be components of a promising vaccine. The optimized conditions of CDAP for the conjugated pneumococal polysaccharide product, of better final quality, were discovered for each of the 1 1 valencies. The conjugates of these pneumococcal polysaccharides obtainable by the improved (optimized) CDAP process, above, (without taking into account the carrier protein, but preferably protein D), are, therefore, a further aspect of the invention.

Example 2 - Study of the Effect of Advanced Adjuvants on the Immunogenicity of the Conjugate Vaccine of PS-PD Pneumococal 1 1 -Valente in Infants Rats The infant rats were immunized with the conjugate vaccine of PS-PD pneumococal 1 1 -valent at a dose of 0.1 μg each polysaccharide (prepared according to the method of Example 1) and using the following adjuvant formulations: none, AIPO4, 3D-MPL, 3D-MPL in AIPO4. The formulation with only 3D-MPL was statistically (and surprisingly) more immunogenic (higher GMC IgG) than the other formulations for 5 out of 1 1 antigens. This was true at both high and low concentrations of 3D-MPL. Opsonophagocytosis confirmed the results of GMC. Materials and Methods Immunization Protocol Infant OFA rats were randomly selected from different mothers and were 7 days old when they received the first immunization. They received 2 additional immunizations 14 and 28 days later. Bleeding was performed on day 56 (28 days after lll). All vaccines were injected s.c. and there were 1 0 rats per vaccine group. The rats were immunized with a 1-valent pneumococal conjugate vaccine comprising the following polysaccharide serotypes conjugated to protein D: 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F.

Formulation To examine the effect of different advanced adjuvants, the dose of the conjugate was kept constant at 0.1 μg of each polysaccharide and adjuvants AIPO4 and 3D-MPL were formulated in different doses and combinations, not including adjuvant at all. These are listed numerically in Table 3 for reference. Adsorption in AIP04 The monovalent adsorbed, concentrated, were prepared according to the following procedure. 50 μg of AIPO (pH 5.1) were mixed with 5 μg of conjugated polysaccharides for 2 hours. The pH was adjusted to pH 5.1 and the mixture was left for an additional 16 hours. 1,500 mM NaCl was added to form the salt concentration up to 150 nM. After 5 minutes, 5 mg / mL of 2-phenoxyethanol was added. After 30 more minutes, the pH was adjusted to 6.1 and left for more than 3 days at 4 ° C. Preparation of diluents Three diluents were prepared in 1 50 mM NaCl / 5 mg / mL phenoxyethanol A: AIPO4 at 1 mg / mL. B: 3D-MP in AIPO4 at 250 and 1000 μg / ml, respectively Weight ratio 3D-MPL / AIPO4 = 5/20 C: 3D-MPL in AIPO4 at 561 and 1000 μg / ml, respectively 3D weight ratio -MPL / AIPO4 = 50/89 Preparation of adsorbed undecactant The eleven monovalent PS-PD, adsorbed, concentrated, were mixed in the correct ratio. The complement of AIPO4 was added as the diluent A. When required, 3D-MPL was added either as an aqueous solution (not adsorbed, Way 1, see below) or as the diluent B or C (3D-MPL adsorbed in AIPO in 2 doses, Way 2, see below). Way 1 3D-MPL was added to the combined adsorbed conjugates as an aqueous suspension. It was mixed with the undecantile for 10 minutes at room temperature and stored at 4 ° C until its administration. Way 2 3D-MPL was pre-adsorbed on AIPO4 before addition to the combined adsorbed conjugates (diluent B and C). To prepare 1 ml of diluent, an aqueous suspension of 3D-MPL (250 or 561 μg) was mixed with 1 mg of AIPO in 1 50 mM NaCl at pH 6.3, for 5 minutes at room temperature. This solution was diluted in NaCl pH 6.1 / phenoxy and incubated overnight at 4 ° C. Preparation of non-adsorbed undecalent The eleven PS-PD conjugates were mixed and diluted in the correct proportion in 1 50 mM NaCl at pH 6.1, phenoxy. When required, 3D-MPL was added as a solution (not adsorbed). The formulations for all injections were prepared 1 8 days after the first administration. ELISA The ELISA was carried out to measure the IgG in rats by using the protocol derived from the WHO Workshop on the ELISA procedure for the quantification of IgG antibody against capsular polysaccharides of Streptococcus pneumoniae in human serum. In essence, the purified capsular polysaccharide is covered directly on the microtiter plate. The serum samples are pre-incubated with the cell wall polysaccharide common to all pneumococci (substance C) and which occurs in ca. 0.5% in purified pneumococcal polysaccharides according to the exposure (EP 72513 B1). Reagents from Jackson Immunolaboratories Inc. were used to detect mouse murine IgG. The titration curves were related to internal standards (monoclonal antibodies) modeled by logarithmic logistic equation. The calculations were carried out by using SoftMax Pro software. The maximum absolute error in these results was expected within a factor of 2. The relative error is less than 30%. Opsonophagocytosis Opsonic titers were determined for serotypes 3, 6B, 7F, 14, 1 9F and 23F by using the CDC protocol (Streptococcus pneumoniae opsonophagocytosis by using Differentiated HL60 cells, Version 1.1) with purified human PMN and baby rabbit complement. The modification included the use of domestic pneumococcal strains and the phagocytic HL60 cells were replaced by purified human neutrophil PMN (there is a high degree of correlation between these phagocytic cells). In addition, 3 mm of glass beads were added to the microtitre cavities to increase the mixture and this allowed the reduction of the proportion of phagocyte: bacteria that was recommended out of 400. Results IgG Concentrations The geometric average IgG concentrations, determined for each serotype and the PD are shown in Tables 4 to . For serotypes 6B, 14, 19F and 23F, the previous results obtained by the use of a tetravalent formulation for comparison are included. The highest IgG concentrations have been highlighted in Tables 4 to 10. The statistical p-value for the 3D-MPL compositions versus the 3D-MPL / AIPO compositions is found in the Table eleven . The adjuvant formulation number 4 (conjugates not adsorbed with a high dose of 3D-MPL) gave the highest GMC for 9 out of 1 1 cases. In 5/1 1 cases, the MPL at the low dose is the second most immunogenic. In addition, adjuvantation gives higher GMC's than when modifying the dose for all serotypes (data not shown) and this is statistically significant for serotypes 4, 6B, 7F, 1 8C and 23F (p <0.05 of 95% Cl) . Opsonophagocytosis The results of opsono tf phagocytosis on deposited serum are shown for serotypes 3, 6B, 7F, 14, 1 9F and 23F in Tables 4 to 8. For the most part, these opsonic titers confirm the GMC IgG. However, the correlation with IgG concentration is greater than 85% for serotypes 6B, 1 9F, 23F (data not shown). For serotype 3, it is important to note that only the 3D-MPL group induced opsonic activity above the threshold. Conclusions In this experiment, it was not expected that the use of 3D-MPL alone induced the highest concentrations of IgG. The maximum IgG of GMC obtained with the modification of the adjuvant was compared with the maximum GMC obtained by modifying the PS dose and it was found that 3D-MPL could induce significantly higher responses in 5/1 1 serotypes. Table 1 1 shows that when comparing 3D-MPL and 3D-MPL / AIPO4 compositions (comparing the formulation process and the 3D-MPL dose), 5 of the polysaccharide conjugates are significantly improved, in terms of immunogenicity, when they are formulated with only 3D-MPL instead of 3D-MPL plus AIPO4: PS 4, PS 6B, PS 1 8C, PS 1 9F and PS 23F. Example 3 - Study of the Effect of the Combination on the Immunogenicity of Conjugates of PS 4, PS 6B, PS 18C, PS 19F and PS 23F in Adult Rats Adult rats were immunized with pneumococal-protein D polysaccharide conjugate vaccines either individually or combined in a multivalent composition (either tetra-, penta-, hepta -or decavalente). Groups of 1 0 rats were immunized twice with a separation of 28 days and blood samples were obtained on day 28 and day 42 (14 days after the 2nd dose). Serum was examined by ELISA for IgG antibodies to pneumococcal polysaccharides. All conjugates induced specific IgG antibodies, as measured by ELISA. The Table 12 shows the effect of the combination of protein D monovalent conjugates of PS 6B, PS 18C, PS 1 9F and PS 23F on their immunogenicity in adult rats, as measured by the concentration of IgG on day 14 after the 2nd dose . A statistical analysis was carried out on all the samples to determine if the differences in the concentration of antibodies after the combination were significant. The combination of any of the protein D conjugates of PS 6B serotypes, PS 18C, PS 19F and PS 23F in a multivalent vaccine did not significantly change its immunogenicity. Table 1 Specific activation / coupling / tempering conditions of conjugates of PS S.pnei / roo ae-Protein D TABLE 2: Specifications of the 11-valent pneumococal PS-PD vaccine (the first numbers of the batch code indicate the seroti or tn OD Table 3. Summary Table of Adjuvant Formulations examined with PS-PD Pneumococal 1 1 -Valente in Infants Rats Table 4. Concentration of Serotype 6B Serotype 6B Serotype, Seroconversion, and Average Opsic IgG Concentration on Day 28 After Immunization III of Infants Rats with PS-PD 11-Valente by the Use of Different Adjuvants and Compound with Tetravalent Immunization) Table 5. Concentration of Serotype 14 Geometric Mean IgG, Seroconversion, and Average Opsic Title on Day 28 After Immunization III of Infant Rats with PS-PD 1 1 -Validating by the Use of Different Adjuvants and Compound with Tetravalent Immunization.

Table 6. Concentration of Serotype 19F Geometric Mediated IgG, Seroconversion and Medium Opsic Title on Day 28 After Immunization III of Infant Rats with PS-PD 1 1 -Validating by the Use of Different Adjuvants and Compound with Tetravalent Immunization Table 7. Middle Genetics IgG Concentration of Serotype 23F, Seroconversion, and Average Opsic Title on Day 28 After Immunization III of Infant Rats with PS-PD 11 -Valente by Using Different Adjuvants and Coming with Tetravalent Immunization Table 8. Mean Geometric IgG Concentration of Serotypes 3 and 7F, Seroconversion and Average Opsic Title on Day 28 After Immunization III of Infant Rats with PS-PD 11-Valente by the Use of Different Adjuvants Table 9. Geometric Mean IgG Concentration of Serotypes 1, 4 and 5, and Seroconversion on Day 28 After Immunization III of Infant Rats Table 10. Geometric Mean IgG Concentration of Serotypes 9V, 18C and PD, and Seroconversion on Day 28 After Immunization of Rats Table 1 1. The statistical significance (p-value) of whether certain pneumococcal polysaccharide conjugates have improved their immunogenicity when formulated with only 3D-MPL versus 3D-MPL / AIPO4. A p-value under 0.01 is considered highly significant. Way 1 and Way 2 indicate the method of formulation.

Table 12. Concentration of Geometric Mean IgG (μg / mL) on day 14 after the 2nd dose after immunization of adult rats with 1.0 μg of polysaccharide-protein D conjugate alone or combined in tetravalent, pentavalent, heptavalent or decavalent These data are combined from 5 separate experiments.

Example 4 - Beneficial impact of the addition of pneumolysin and 3D-MPL on the protective efficacy of the 1-polysaccharide vaccine -valent conjugated with PD against pneumococal lung colonization in mice Immunological readings Serum IgG ELISA dose of pneumolysin specific Immunoplates of Maxisorp Nunc were covered for 2 hours at 37 ° C with 100 μl / well of 2 μg / ml of recombinant native pneumolysin (PLY) diluted in PBS. The plates were rinsed 3 times with 0.9% NaCl and 0.05% Tween-20 regulator. Then, 2-fold serial dilutions (in PBS / 0.05% Tween 20, 100 μl per well) of an anti-PLY serum reference were added as a standard curve (starting at 670 ng / ml IgG) and samples from serum (starting at a dilution of 1/10) were incubated for 30 minutes at 20 ° C under agitation. After rinsing horn and described previously, goat anti-mouse IgG conjugated with peroxidase (Jackson) diluted 5000x in PBS / 0.05% Tween 20, was incubated (100 μl / well) for 30 minutes at 20 ° C under agitation. After rinsing, the plates were incubated for 15 minutes at room temperature with 100 μl / well of revelation buffer (OPDA 0.4 mg / ml and 0.05% H2O2 in 100 mM pH 4.5 citrate buffer). The revelation was stopped by adding 50 μl / 1 N cavity of HCl. The optical densities were read at 490 and 620 nm by the use of an Emax immunolector (Molecular Devices). The antibody titer was calculated by the 4-parameter mathematical method using SoftMaxPro software.

Inhibition of Hemolysis This assay was done to measure the ability of serum antibodies to inhibit the hemolytic activity of pneumolysin (PLY). In order to eliminate cholesterol (susceptible to interact with PLY), the serum samples were treated 2x as follows: they were mixed with an equal volume of chloroform and then incubated for 45 minutes under agitation. The supernatants were collected after centrifugation for 10 minutes at 1000 rpm. Cholesterol clarified serum was diluted (2-fold serial dilutions in 1 mM dithiothreitol, 0.01% BSA, 15 mM TRIS, 150 mM NaCl, pH 7.5) in 96-well microplates (Nunc). 50 μl of a solution containing 4 HU (Hemolysis Units) of PLY in each well was added and incubated for 15 minutes at 37 ° C. Then, 100 μl of goat red blood cells (1% solution) was added for 30 minutes at 37 ° C. After centrifugation for 10 minutes at 1000 rpm, the supernatants (150 μl) were collected and placed in another 96-well microplate for optical density reading at 405 nm. The results were expressed as half-point dilution titers. Chemical detoxification of pneumolysin Recombinant native pneumolysin (PLY) was dialyzed against Phosphate, 50 mM NaCl, 500 mM pH 7.6 regulator. All subsequent steps were made at 39.5 ° C under episodic agitation. On day 1, 10% Tween-80 (1/250 v / v), 57.4 mM N-acetyl tryptophan pH 7.6 (3/100 v / v), 2.2 M glycine in buffer were added to a PLY solution. Phosphate (1/100 v / v) and 10% formaldehyde in Phosphate buffer (3/100 v / v).

On days 2 and 3, 1 0% formaldehyde was added again, at a ratio of 3/1 00 and 2/100 v / v, respectively. Incubation at 39.5 ° C was sustained until day 7 under episodic agitation. Finally, PLY was dialyzed against 50 mM Phosphate, 500 mM NaCl pH 7.6 regulator. Complete inactivation of PLY was demonstrated in the hemolysis assay. Pneumococal intranasal stimulation in OF1 mice of seven-week-old OF1 female mice was inoculated intranasally under anesthesia with 5,105 CFU of S. pneumoniae serotype 6B adapted to mouse. The lungs were removed 6 hours after stimulation and homogenized (Ultramax, 24000 rpm, 4 ° C) in a medium of Todd Hewith Broth (THB, Gibco). Serial 10-fold dilutions of lung homogenates were plated overnight at 37 ° C on Petri dishes containing THA juice supplemented with ferment extract. The pneumococal lung infection was determined as the number of CFU / mouse, expressed as average logarithmic weight. The limit of detection was 2.14 log CFU / mouse. Example 4A Adjuvant effect of 3D-MPL on the immune response of anti-pneumolysin In the present example, we evaluated the impact of the adjuvantation of 3D-MPL on the immune response to recombinant native pneumolysin (PLY, provided by J. Paton, Children's Hospital, North Adelaide, Australia) and its chemically detoxified counterpart (DPLY). The chemical detoxification was done as described above. Groups of 10 6-week-old females of Balb / c mice were immunized intramuscularly on days 0, 14 and 21 with 1 μg of PLY or DPLY contained in either A: 100 μg AIPO4; or B: AIPO4 1 00 μg + 5 μg of 3D-MPL (3-de-O-acylated monophosphoryl lipid A, provided by Ribi Immunochem). Figures 1A and 1B show ELISA IgG and HemoLisis inhibition titers (HLI) measured in poWder serum. Whatever the antigen, the best immune responses were induced in animals vaccinated with formulations supplemented with 3D-MPL. Interestingly, DPLY was as immunogenic as PLY when administered with AIPO4 + 3D-MPL, although it was a weaker immunogen in the AIPO4 formulation. This showed the advantageous ability of 3D-MPL to improve the antibody response to detoxified pneumolysin. In compositions containing pneumolysin, it may be preferable to use chemically detoxified pneumoiisin instead of mutationally detoxified pneumolysin. This is because the detoxified mutants obtained to date still have residual toxin acti - the chemically detoxified pneumolysin does not. Accordingly, it is considered another general aspect of the invention that, in general, compositions comprising pneumolysin (or pneumolysin mutants) that have been chemically detoxified for use in a vaccine, should be adjuvanted with a Th 1 adjuvant, preferably 3D- MPL. Such compositions are provided by the invention. Also provided is a method for increasing the immune response of chemically detoxified pneumolysin within an immunogenic composition comprising the steps of adding a Th 1 adjuvant (preferably, 3D-MPL) to the composition. Example 4B Beneficial impact of the addition of an attenuated mutant of pneumolysin and 3D-MPL adjuvant on the protective efficacy of polysaccharide vaccine 1 -valent conjugated with PD against lung pneumococal colonization in OF1 mice stimulated intranasally with serotype 6B . In the present example, we evaluated the prophylactic efficacy of a vaccine containing the conjugate of polysaccharide 1 1 -valent-protein D, the attenuated mutant pneumolysin antigen (PdB, WO 90/06951) and adjuvants of AIPO4 + 3D-MPL, in comparison with the classical conjugate formulation of polysaccharide 1 1 -valent-protein D adsorbed on AIPO4. Groups of 12 four-week-old OF1 female mice were immunized subcutaneously on days 0 and 14 with formulations containing A: 50 μg of AIPO4; B: 0.1 μg of PS / serotype of polysaccharide vaccine 1 1 -valent conjugated with PD + 50 μg of AIPO4; or C: 0.1 μg PS / serotype of 1-polysaccharide vaccine -valent conjugated with PD + 10 μg of PdB (provided by J. Patón, Children's Hospital, North Adelaide, Australia) + 50 μg of AIPO4 + 5 μg of 3D- MPL (supplied by Ribi Immunochem). The stimulation was done on day 21 as described above. As shown in Figure 1C, a very significant protection (p <0.007) was given by the conjugate vaccine polysaccharide 1 -valent complemented with PdB and adjuvanted with AIPO4 + MPL (the black bars represent the arithmetic method). On the contrary, no significant protection was observed in animals immunized with the formulation of the conjugate of polysaccharide 1 -valent / AIPO4. This result proved that the addition of pneumolysin antigen (still attenuated) and adjuvant of 3D-MPL, improved the effectiveness of the polysaccharide conjugate vaccine 1 1 -valent against pneumonia. Example 4C, Immune Correlations of the Protection Shown in Example 4B In order to establish the immune protection correlations granted in Example 4B, by conjugate polysaccharide vaccine 1 -valenté supplemented with attenuated mutant pneumolysin (PdB) and 3D-lPL, anti-serological responses prior to stimulation with respect to polysaccharide 6B and PdB were measured. Antibody titers were then compared with numbers of colonies and bacteria measured in the lungs of the corresponding animals at 6 hours after stimulation. R2 was calculated on linear regressions of Log / Log. R calculated was equal to 0.18 and 0.02 for anti-PdD and anti-6B antibody responses, respectively. This shows the absence of correlation between the humoral immune responses and the protection of both of them. The anti-6B antibody titers were not significantly different in the groups immunized with the conjugate vaccine 1-v? | Lens (GMT = 0.318 ng / ml) or with the same vaccine supplemented with PdC and 3D-MPL (GMT = 0.458 ng / ml) Therefore, the evaluation. Additional groups of mice were sacrificed on day 7 and sampled according to the above. Results: The figures in the parentheses are numbers of animals that died before the sampling time. Conclusion: In general, there was no significant difference in the bacterial numbers isolated from any of the treatment groups. This indicates that no measurable protection was provided by the anti-polysaccharide at concentrations up to and including 5 μg / ml. This is similar to what was observed in some human clinical trials, that is, the polysaccharide antibody is insufficient for protection against pneumococcal pneumonia in some populations. Example 5B - Determine pneumonia protection provided by the active administration of Ply (pneumolysin) with or without adjuvant and synergy with sub-optimal anti-PS antibody. Method Animals: 128 male CD-1 mice (6 weeks of age on immunization, 10 weeks of age on infection) from Charles River, St. Constant, Quebec, Canada. The animals weighed approximately 20 gm at 6 weeks and 38 g at 10 weeks. Immunizations: Six groups of 16 mice were immunized by subcutaneous injection on days -22 and -14 with 100 ul of vaccine as detailed below. (128 mice in total). The PdB (WO 90/06951) was obtained as a courtesy of Dr. James Patón, Australia. 3D-MPL was obtained from Ribi / Corixa. On day -1, specific groups were immunized (see Table below) (ip 100 μl) passively with a concentration of 4.26 μg / ml (4 ml of 5 μg / ml + 1.3 ml of 2 μg / ml). mouse anti-polysaccharide antibody.

Infection: On day 0, the mice were anesthetized (3% isoflurane plus 1 L / min O2). Bacterial inoculation was prepared by harvesting the development of S. pneumoniae N1387 (serotype 6) from trypticase soy agar plates (TSA) supplemented with 5% equine blood and suspended in 6 ml of PBS. A ten-fold dilution (1 ml plus 9 ml) was prepared on cooled ground nutrient agar (maintained at 41 ° C) immediately before infection. Mice were infected by intra-bronchial instillation through intra-tracheal intubation and received approximately 6.0 log 10 cfu / mouse in a volume of 50 μl. This method was described by Woodnut and Berry (Antimicrob, Ag. Chemotherap, 43:29 (1999)). Samples: At 72 hours after infection, 8 mice / group were sacrificed by CO2 overdose and the lungs were removed and homogenized in 1 ml of PBS. Ten-fold serial dilutions in PBS were prepared to enumerate viable bacterial numbers. Samples were inoculated (20 μl) in triplicate in TSA plates supplemented with 5% equine blood and incubated overnight at 37 ° C before evaluation. Additional groups of mice were sacrificed on day 8 after infection and sampled as above. Data Analysis The resulting measurement for comparison of treatment was the number of bacteria in the lungs on day 3 and 6 after infection. The results are presented as group means with standard deviations. The statistical analysis was carried out by using Student's t test, where a P value of < 0.05 was considered insignificant. Results: 72h after infection Bacterial counts of group 1-4 were significantly lower (p <0.05) than those of group 1 -3. Bacterial counts of group 1-4 were significantly lower (p <0.05) than those of group 1 -5. 168h after infection The bacterial numbers in all groups were approximately 2 logs less than 8 days than at 3 days, indicating that the infection was being resolved. The bacterial counts of group 1 -2 were significantly lower (p <0.05) than those of group 1 -5.

As shown above, the anti-polysaccharide antibody alone (group 1 -5) does not provide protection against the development of pneumococci in pu imon. PdB adjuvanted with AIPO4 does not grant protection either, but on day 8 there is a tendency to protection when PdB is combined with 3D-MPL (group 1 -2). On day 3, the most significantly protected group, group 1-4, had all three elements, PdB, 3D-MPL and anti-polysaccharide antibody administered passively. This conclusion is supported by the rate of mortality. The group 1 -4 had only 2/8 deaths compared to 5/10 for groups 1 -5 and 1 -3.

Conclusion: Since the experiment was carried out with passively immunized animals, the synergistic effect of also actively immunising with pneumolysin and MPL can not be due to an increase in the level of antibodies against the polysaccharide antigen. Since the animals were only passively immunized against the pneumococal polysaccharide, by day 8 the levels of such an antibody would have largely dissipated from the host. Even, significant protection against pneumococal pneumonia could be observed in groups immunized with pneumolysin plus 3D-MPL and especially in groups immunized with pneumolysin plus 3D-MPL plus passively administered anti-polysaccharide antibody, indicating the synergy of this combination. If the anti-polysaccharide immunization had been carried out actively (preferably with conjugated polysaccharide), the effect would have been even more marked, since the effect of the B cell memory and the constant levels of anti-PS antibody would have contributed to the cooperation of the immune response (see, for example, Figure 1 C where many of the animals immunized actively with polysaccharide and protein demonstrated no bacteria in the lungs after stimulation). Example 6 - Immunogenicity in 1-year-old Balb / C mice of pneumococal 1 1 -valent polysaccharide conjugate vaccine - Protein D, adjuvanted with 3D-MPL Introduction and Oberant (s): Protection against pneumococal infection is mediated by serotype-specific antibodies through opsonophagocytosis. It can be assumed that increases in the concentration of the antibody will result in increased protection and, consequently, much effort has been expended in finding ways to increase the humoral response. One strategy that has been successfully applied to conjugate vaccines in pre-clinical studies is the use of immunostimulatory adjuvants (reviewed in Poolman et al 1998, Carbohydrate-Based Bacterial Vaccines, In: Handbook of Experimental Pharmacology, Eds. P. Perlmann and H. Wigsell, Springer-Verlag, Heidelberg, D). The data presented in this section show the results of the latest experiments using clinical batches in a protocol designed to mimic a clinical trial. Protocol: One-year-old balb / c mice were immunized with 1/10 of the human dose of pneumococcal polysaccharide-protein D conjugate vaccine or 23-valent full polysaccharide vaccine. The vaccines used were clinical lots DSP009, DSO013 or DSP014, corresponding to the dose of 1 mcg of serotypes 6B and 23F and 5 mcg of the remaining serotypes of the conjugate vaccine 1 1 -valent, the dose of 0.1 mcg of the conjugate vaccine 1 1 -valent or the dose of 0.1 mcg of conjugate vaccine 1 1 -valent adjuvanted with 5 mcg of 3D-MPL, respectively. All 1 1 -valent conjugate vaccines were also adjuvanted with 50 μg of AIPO4. Groups of 20 mice were immunized intramuscularly. The injections of the groups listed in the following table were carried out on days 0 and 21. The test bleeds were obtained on day 35, (14 days after the second dose).

Table: Immunization Program for 1-year-old Balb / c mice with clinical lots of pneumococalotein D polysaccharide conjugate vaccine.

The serum was examined by ELISA for IgG antibodies to the pneumococcal polysaccharides following the consensus protocol of CDC / WHO, ie, after neutralization of the serum with cell wall polysaccharide. The ELISA was calibrated to give antibody concentrations in mcg / ml by the use of serotype-specific IgG1 monoclonal antibodies. The statistical analyzes of comparisons were calculated by using UNISTAT version 5.0 beta. ANOVA by the Tukey-HSD method was carried out in IgG concentrations transformed by logarithm. The similar comparison to pairs of seroconversion speeds was carried out by using an exact test of Fisher. Results: The GMC IgG and the 95% confidence interval against the 1 1 serotypes and D protein induced 14 days after the second immunization (dose 2) are shown in the following table. The seroconversion speeds are shown where an interval of 95% confidential Group 1 shows the effect of immunization with simple polysaccharides, which normally induce only IgM in animals. Most IgG levels are below the detection threshold; however, the balb / c mice were able to elaborate IgG for a few pneumococcal polysaccharides, notably serotypes 3, 19F and 14. Immunization with conjugate vaccines induced antibody from IgG with high seroconversion rates against all serotypes except 23F. A dose-dependent response (group 4 vs group 2) was observed only for serotypes 7F and 19F, but these observations were not statistically significant. A greater response was observed after two doses (groups b vs groups a) for the serotypes 3, 6B, 7F and 19F and PD and these observations were statistically significant in many cases with the 3 formulations. The most interesting is the effect of 3D-MPL. Two doses of the vaccine formulated with 3D-MPL (group 3b) induced the highest GMC of specific IgG and this was statistically significant for all serotypes except 23F, in which case there was a significantly higher seroconversion rate (p = 0.02 group 3b vs 2b, Fisher's exact).

Table: Geometric Mean [IgG] and 95% Confidence Intervals for Selected Pneumococal Serotypes and Protein D in Balb / c 1 year old, 14 Days After Immunization II with Conjugate Vaccine standard above the average of the negative control. Please refer to the previous table for group definitions. Conclusion: The data presented here demonstrate that the addition of 3D-MPL to the conjugate vaccine of pneumococal polysaccharide 1 1 -valent-Protein D increased the immune response in aged balb / c mice for all serotypes examined. In most cases, two doses of vaccine induced geometric mean IgG concentrations greater than one dose. Since this is not observed when using simple polysaccharide vaccine, even in humans, it is considered an indication of a T cell-dependent immune response and the induction of immune memory. These data support a vaccine administration scheme by the use of pneumococcal polysaccharides adjuvanted with Th 1 adjuvants (preferably 3D-MPL), whereby at least two doses of the adjuvanted vaccine are administered, preferably 1-2 separate pieces and more preferably 3 weeks apart. Such administration program is considered a further aspect of the invention. The mice used in the experiment did not respond to PS 23 (simple or conjugated). Interestingly, although antibody levels against the polysaccharide remained with low consideration to the vaccine composition used, many more mice responded to PS 23 when 3D-MPL was used as the adjuvant (seroconversion being significantly greater). A use of Th1 adjuvants, particularly 3D-MPL, in vaccine compositions comprising conjugated pneumococcal polysaccharides in order to alleviate the inability to respond to a pneumococal polysaccharide in a vaccine, is still a further aspect of the invention. A method for alleviating the inability to respond with the aforementioned composition by using the two administration schemes described above is still another aspect. Example 7 - Conjugate of Polysaccharide C - Neisseria Meningitidis Protein D (PSC-PD) A: EXPRESSION OF PROTEIN D As in Example 1. B: ELABORATION OF POLYSACCHARIDE C The source of the polysaccharide of group C is the C1 1 strain of N. meningitidis. This is fermented by the use of classical fermentation techniques (EP 72513). The dry powder polysaccharides used in the conjugation process are identical to Mencevax (SB Biologicals S.A.). An aliquot of strain C1 1 thaws and 0.1 ml of solution are placed on a Petri dish of Mueller Hinton medium, supplemented with ferrous extract dialysis (10%, v / v) and incubated for 23 to 25 hours at 36 ° C in an air incubator saturated with water. The development of the surface is then suspended again in a sterilized fermentation medium and inoculated with this suspension in a Roux bottle containing Mueller Hinton medium supplemented with dialysate of ferment extract (10%, v / v) and glass globules sterile After incubation of the Roux bottle for 23 to 25 hours at 36 ° C in an air incubator saturated with water, the surface development is resuspended in 10 ml of sterile fermentation medium and 0.2 to 0.3 ml of this suspension is inoculated into another 12 Roux bottles of Mueller Hinton medium. After incubation for 23 to 25 hrs at 36 ° C in an air incubator saturated with water, the surface development is resuspended in 10 ml of sterile fermentation medium. The bacterial suspension is deposited in a conical flask. This suspension is then transferred aseptically to the fermentor by the use of sterile syringes. The fermentation of the meningococcus takes place in fermenters contained in a clean room, under negative pressure.

The fermentation is generally completed after 10-12 hours corresponding to approximately 1010 bacteria / ml (ie, the early stationary phase) and is detected by increasing pH. In this stage, the whole fermentation broth is inactivated by heat (12 minutes at 56 ° C) before centrifugation. Before and after the inactivation, a sample of the broth is taken and placed on petri dishes of Mueller Hinton medium. C: PURIFICATION OF PS The purification process is a multistage process carried out over the entire fermentation broth. In the first purification step, the inactivated culture is clarified by centrifugation and the supernatant is recovered. The purification of the polysaccharide is based on precipitation with a quaternary ammonium salt (cetyltrimethylammonium bromide / C , CETAVLON R). CTAB forms insoluble complexes with polyanions such as polysaccharides, nucleic acid and proteins that depend on their pl. Following ionic controlled conditions, this method can be used to precipitate impurities (low conductivity) or polysaccharides (high conductivity). The polysaccharides included in the clarified supernatant are precipitated by the use of diatomaceous earth (CELITER 545) as matrix, to avoid the formation of inert insoluble mass during the different precipitations / purifications. Purification scheme for N.menengitidis C polysaccharide: Stage 1: Fixation of the PSC-CTAB complex in CELITER 545 and removal of cell debris, nucleic acids and proteins by rinsing with 0.05% C . Stage 2: Levigation of PS with 50% EtOH. The first fractions that are turbid and contain impurities and LPS are discarded. The presence of PS in the following fractions is verified by obstruction test. Stage 3: Refixation of the PS-CTAB complex in CELITER 545 and removal of nucleic acids and smaller proteins by rinsing the 0.05% C . Stage 4: Levigation of PS with 50% EtOH. The first turbid fractions are discarded. The presence of PS in the following fractions is verified by obstruction test. The levigado is filtered and the levigado containing the crude polysaccharide is collected. The polysaccharide is precipitated from the filtrate by the addition of ethanol to a final concentration of 80%. The polysaccharide is then recovered as a white powder, dried by vacuum spray and stored at -20 ° C. D: CONJUGATION OF CDAP Conjugation of PSC and PD For the conjugation of PSC and PD, the technology of CDAP conjugation to classical activation of CNBr and the coupling through a separator to the carrier protein is preferred. The polysaccharide is first activated by cyanoylation with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). CDAP is a water-soluble cyanation reagent in which the electrophilicity of the cyano group is increased over that of CNBr, allowing the cyanolation reaction to be carried out under relatively mild conditions. After activation, the polysaccharide can be coupled directly to the carrier protein through its amino groups without the introduction of any spacer molecules.

The unreacted estercyanate groups are annealed by extensive reaction with glycine. The total number of stages involved in the preparation of the conjugate vaccines is reduced and, more importantly, the potentially immunogenic spreader molecules do not occur in the final product. Activation of polysaccharides with CDAP introduces a cyanate group into the polysaccharides and releases dimethylaminopyridine (DMAP). The cyano group reacts with NH2 groups on the protein during the subsequent coupling procedure and becomes a carbamate. Activation of PSC and coupling of PSC-PD Activation and coupling are carried out at + 25 ° C. 120 mg of PS are dissolved for at least 4 hours in WFI. The CDAP solution (100 mg / ml recently prepared in acetonitrile) is added to reach a CDAP / PS (w / w) ratio of 0. 75. After 1 min 30, the pH is raised to the activation pH (pH 10) by the addition of triethylamine and stabilized until the addition of PD. At 3 min 30, NaCl is added to a final concentration of 2M. At the time of 4 min, the purified PD is added to reach a PD / PS ratio of 1.5 / 1; the pH is immediately adjusted to the coupling pH (pH 10). The solution is left for 1 hour under pH regulation. Tempering 6 ml of a 2M solution of glycine is added to the PS / PD, CDAP mixture. The pH is adjusted to the tempering pH (pH 8.8). The solution is stirred for 30 minutes at the working temperature, then overnight at + 2-8 ° C with continuous slow stirring. Purification of PS-PD After filtration (5 μm), the PS-PD conjugate is purified in a cold room by gel permeation chromatography on a S400HR Sephacryl gel to remove small molecules (including DMAP) and unconjugated PD: Levigation - 150 mM NaCl pH 6. 5; Monitoring - UV 280 nm, pH and conductivity. Based on the different molecular size of the reaction components, the PS-PD conjugates are first levigated followed by free PD and finally DMAP. The fractions containing the conjugate are deposited, as detected by DMAB (PS) and μBCA (protein). The deposited fractions are filtered sterile (0.2 μm). E: ADJUSTED CONJUGATED VACCINE FORMULATION IN PSC-PD AIP04 rinse In order to optimize the adsorption of PSC-PD conjugate in AIPO, AIPO4 is rinsed to reduce the concentration of PO4"3: AIPO is rinsed with 50 mM NaCl 1 and centrifuged (4x), the pellet is then resuspended in 150 mM NaCl, then filtered (100 μm), and the filtrate is heat sterilized.This AIPO4 is referred to as WAP (phosphate rinsed in autoclave), Fumigation The volume of the PSC-PD conjugate is adsorbed in WAP of AIPO4 before the final formulation of the finished product. The WIPO of AIPO was stirred with with PSC-PD for 5 minutes at room temperature. The pH was adjusted to 5.1, and the mixture was stirred for a further 18 hours at room temperature. The NaCl solution was added at 150 mM and the mixture was stirred for 5 minutes at room temperature. 2-Phenoxyethanol was added at 5 mg / mL and the mixture was stirred for 15 minutes at room temperature, then adjusted to pH 6.1. Composition / final dose PSC-PD: 10 μg of PS-WAP of AIPO4: 0.25 mg of Al3 + NaCl: 150 mM 2-phenoxy-ethanol: 2.5 mg Water for injection: up to 0.5 ml pH: 6.1 F: PRECLINICAL INFORMATION Immunogenicity polysaccharide conjugate in mice The immunogenicity of the PSC-PD conjugate has been determined in Balb / c mice from 6 to 8 weeks of age. The simple conjugate (not adsorbed) or the conjugate adsorbed on AIPO4 was injected as a monovalent vaccine. The anti-PSC antibodies induced were measured by ELISA while the functional antibodies were analyzed by the use of the bactericidal test, both methods being based on the protocols of the CDC (Centers for Disease Control and Prevention, Atlanta, USA). We present the results of two different experiments carried out to determine the response versus the effect of dose and adjuvant (AIPO4). Dose-range experiment In this experiment, PSC-PD was injected twice (two weeks apart) in Balb / C mice. Four different doses of conjugate formulated in AIPO4 were used: 0.1; 0.5; 2.5; and 9.6 μg / animal. Mice (1 O / group) were bled on day 14 (14 after I), 28 (14 after II) and 42 (28 after II). The geometric mean concentrations (GMCs) of polysaccharide C specific antibodies measured by ELISA were expressed in μg IgG / ml by the use of purified IgG as a reference. The bactericidal antibodies were measured in deposited serum and titres expressed as the reciprocal of the dilution capable of eliminating 50% of bacteria, using the C1 1 strain of N. meningitidis in the presence of baby rabbit complement. The response to the dose obtained showed a plateau from the 2.5 μg dose. The results indicate that there is a good response to reinforcement between day 14 after I and 14 after II. Antibody levels on day 28 after II are at least equivalent to those on day 14 after II. Bactericidal antibody titers are found according to ELISA concentrations and confirm the immunogenicity of the PSC-PD conjugate. Effect of the Adjuvant In this experiment, a batch of PSC-PD conjugate formulated in AIPO4 was determined, the simple conjugate (not adjuvanted) was injected for comparison. 10 mice / group were injected twice, with a separation of two weeks, by the subcutaneous route, with 2 μg of conjugate. Mice were bled on day 14 (14 after I), 28 (14 after II) and 42 (28 after II) and ELIS and functional antibody titers were measured (only on day 14 after the II and 28 after the II for the bactericidal test). The AIPO4 formulation induces antibody titers up to 10 times higher compared to non-adjuvanted formulations. Conclusions The following general conclusions can be made from the results of the experiments described above: - The PSC-PD conjugate induces an anamnestic response demonstrating that when PSC is conjugated, it becomes a T cell-dependent antigen. anti-PSC measured by ELISA correlate well with the bactericidal antibody titers showing the antibodies induced by the PSC-PD conjugate are functional against serogroup C of N. meningitidis. Approximately 2.5 μg of conjugate adsorbed on AIPO4 seems to produce an optimal antibody response in the mice. The chemistry of CDAP seems to be an adequate method to make the immunogenic PSC-PD conjugates. Example 8 - Preparation of a Serogroup A Conjugate Polysaccharide from N.meningitidis - PD A dry powder of polysaccharide A (PSA) is dissolved for one hour in a 0.2 M NaCl solution at a final concentration of 8 mg / ml. The pH is then set at a value of 6 with either HCl or NaOH and the solution is thermoregulated at 25 ° C. 0.75 mg of CDAP / mg of PSA (a preparation at 100 mg / ml of acetonitrile) is added to the PSA solution. After 1.5 minutes without pH regulation, 0.2 M NaOH is added to obtain a pH of 10. 2.5 minutes later, protein D (concentrated at 5 mg / ml) is added according to a ratio of PD / PSA of approximately 1. A pH of 10 is maintained during the 1 hour coupling reaction period. Then, 10 mg of glycine (2 M pH 9.0) / mg of PSA are added and the pH is adjusted to a value of 9.0 for 30 minutes at 25 ° C. The mixture is then stored overnight at 4 ° C before purification by exclusion column chromatography (Sephacryl S400HR from Pharmacia). The conjugate is first reacted, followed by unreacted PD and by-product (DMAP, glycine, and salts). The conjugate is collected and sterilized by a 0.2 μm filtration in a Sartorius Sartopore membrane. Example 9 - In vitro characterizations of the products of Examples 7 and 8 The main characteristics are summarized in the table below: In Vivo Results Balb / C mice were used as an animal model to test the immunogenicity of the conjugates. The conjugates were adsorbed either in AIPO or AI (OH) 3 (10 μg of PS in 500 μg of Al3 +) or not absorbed. The mice were injected as follows: 2 injections at two week intervals (2 μg PS / injection). From these results, we can conclude first that free PS greatly influences the immune response. Conjugates having less than 10% free PS have obtained better results. The improvements prior to the CDAP process are therefore a further aspect of the invention. The formulation is also important. AIPO4 seems to be the most appropriate adjuvant in this model. The conjugates induce a reinforcing effect that is not observed when the polysaccharides are injected alone. Conclusions The conjugates of N. meningitidis A and C were obtained on a final PS / protein ratio of 1 and 0.6-0.7 (w / w), respectively. The free PS and its free vehicle protein were below 10% and 15%, respectively. The polysaccharide recovery is greater than 70%. The PSA and PSC conjugates obtainable by the improved CDAP process (optimized), (without taking into account the vehicle protein, but preferably protein D) are, therefore, an additional aspect of the invention. Example 10 - Preparation of a Polysaccharide Conjugate of H. influenzae b - PD H. influenzae b is one of the leading causes of meningitis in children less than 2 years of age. The capsular polysaccharide of H. influenzae (PRP) as a conjugate on tetanus toxoid is well known (conjugated by chemistry developed by J. Robbins). CDAP is an improved chemistry. The following is the count of the optimal conditions of CDAP found in the conjugation of PRP, preferably to PD. The parameters that influence the reaction of the conjugation are the following: The initial concentration of polysaccharide (which can have a double impact on the final levels of free polysaccharide and in the sterile filtration stage). The initial concentration of the vehicle protein.

The initial ratio of polysaccharide to protein (which can also have double impact on the final levels of free polysaccharide and in the sterile filtration stage). The amount of CDAP used (usually in large excess). The temperature of the reaction (which can influence the breakdown of the polysaccharide, the kinetics of the reaction and the breakdown of the reactive groups). The pH of activation and coupling. The tempering pH (which includes the residual DMAP level). The activation, coupling and tempering time. The present inventors have found that the 3 most critical parameters to optimize the quality of the final product are: the initial polysaccharide / protein ratio; the initial concentration of polysaccharide; and the coupling pH. A reaction cube with the 3 previous conditions was designed like the three axes. The central points (and range of experienced value) for these axes are: PS / protein ratio - 1/1 (+ 0.3 / 1); [PS] = 5 mg / ml (+2 mg / ml); and coupling pH = 8.0 (+1 .0 pH unit). The less essential parameters were fixed in the following: 30 mg of polysaccharide were used; temperature 25 ° C; [CDAP] = 0.75 mg / mg PS; pH titrated with 0.2M NaOH; Activation pH = 9.5; temperature for activation = 1.5 minutes; coupling temperature - 1 hour; [protein] = 10 mg / ml; Tempering pH = 9.0; tempering temperature = 1 hour; dissolution temperature of PS in solvent = 1 hour in 2M NaCl; purification on Sephacryl S-400HR levigated with NaCl 1 50 mM at 12 cm / hour; and filter sterilization with a SARTOLAB P20 at 5 ml / min. The data that sought to establish optimized conditions when producing products within the aforementioned reaction cube were: process data - maximum production after filtration, maximum level of protein incorporated; and quality of the product data - final ratio of PS / protein, level of free PS, level of free protein, minimum levels of residual DMAP (a product of CDAP fracture). Filtration Result The factor that affects the result after filtration is the interaction between the initial [PS] and the coupling pH and the initial PS / protein ratio. At low [PS], there is little interaction with the last two factors and a good filtration capacity always results (approximately 95% for all products). However, at high concentrations the filtration capacity decreases if the pH and the initial proportion increase (high PS, very small proportion, very low pH = 99% filtration, but high PS, maximum proportion and pH = 19% filtration). Level of protein incorporation The proportion of the final proportion of PS / protein with respect to the initial ratio is a measure of coupling efficiency. At a high [PS], the pH does not effect the proportion of the proportions. However, the initial proportion does (1 .75 at low initial proportion, 1 .26 at high initial proportions). A low [PS], the proportion of the proportions is lower for most, however, the pH now has more effect (low pH, low ratio = 0.96, low pH, high proportion = 0.8, high pH, low ratio = 1.4, and high pH, high proportion = 0.92). Final proportion of PS / protein The final proportion depends on the initial ratio and the [PS]. The most dimensionable final proportions are obtained with a combination of high initial proportions and high [PS]. The effect of pH on the final proportion is not as significant as a weak [PS].

Free protein D level Minor amounts of free protein D are observed at high pH and high [PS] levels (levels approaching 0.0). The effect of high [PS] becomes especially marked when the pH is low. The elevation of the initial ratio contributes rather little to the increase in free D protein. Residual DMAP The initial ratio does not have a significant effect. In contrast, the level of DMAP increases with [PS] and decreases when the pH rises. Conclusions The most preferred conjugation conditions are therefore the following: coupling pH = 9.0; [PS] = 3 mg / ml; and initial ratio = 1/1. With such conditions, the characteristics of the final product are as follows: The PRP conjugates obtainable by the enhanced (optimized) CDAP process above (without taking into account the carrier protein, but preferably D protein) are therefore a further aspect of the invention. Example 11: Protein D as an antigen - the way in which its protective efficacy against non-typeable H. influenzae can be improved by its formulation with 3D-MPL Female Balb / c mice (10 per group) were immunized (intramuscularly) with the conjugate pneumococcal polysaccharide vaccine undecalent - protein D for the first time at the age of 20 weeks (DO) and receive a second immunization two weeks later (D14). The blood is collected 7 days after the second immunization. The antibody titers against protein D were measured in terms of the amount of IgG1, IgG2a and IgG2b antibodies. Lyophilized undecalent vaccines (without AIPO) were prepared by combining the conjugates with 15.75% lactose, shaking for 15 minutes at room temperature, adjusting the pH to 6.1 + 0.1, and lyophilization (the cycle usually starts at -69 ° C , adjusting gradually to -24 ° C for 3 hours, then this temperature is retained for 18 hours, gradually adjusting to -16 ° C for 1 hour, then retaining this temperature for 6 hours, then gradually adjusting to + 34 ° C for 3 hours. hours and finally retaining this temperature for 9 hours). The composition of the formulations and the reconstituents for lyophilisates are presented in Table 1 3. The most characteristic measurement as to whether the Th1-type cell-mediated immune response has occurred, is known to correlate with the level of IgG2a antibody . As can be seen from the data, a surprisingly large increase in IgG2a results if protein D has been lyophilized with a Th1 adjuvant (in this case, 3D-MPL).

Table 13: Formulation composition (by human dose) and antibody titers against protein D in mice (with 1/10 dose) before the injection; +/- 2 hours before injection; 2-phenoxyethanol

Claims (1)

CLAIMS 1. An immunogenic composition, characterized in that it comprises at least one polysaccharide antigen of Streptococcus pneumoniae, at least one protein antigen of Streptococcus pneumoniae or immunologically functional equivalent thereof and an adjuvant, which is a preferential inducer of a TH1 response. 2. The immunogenic composition according to claim 1, characterized in that the protein antigen is an outer surface protein or a protein secreted from Streptococcus pneumoniae or immunologically functional equivalents thereof. 3. The immunogenic composition according to claims 1 or 2, characterized in that the protein antigen is a toxin, adhesin or lipoprotein of Streptococcus pneumoniae or immunologically functional equivalents thereof. 4. The immunogenic composition according to the claims

1 - . 1-3, characterized in that the protein antigen or immunologically functional equivalent thereof is selected from the group: pneumolysin, PspA or variants of transmembrane omission thereof, PspC or variants of transmembrane omission thereof, PsaA or variants of transmembrane omission thereof, glyceraldehyde-3-phosphate dehydrogenase and CbpA or variants of transmembrane omission thereof. The immunogenic composition according to claims 1-4, characterized in that the polysaccharide antigen occurs in the form of a polysaccharide-protein carrier conjugate. 6. The immunogenic composition according to claim 5, characterized in that the carrier protein is selected from the group consisting of: Diphtheria toxoid, Tetanus toxoid, CRM197, Bocallave Limpet hemocyanin (KLH), Tuberculin protein derivative (PPD) ) and protein D from H. influenzae. An immunogenic composition according to any of claims 1 to 6, characterized in that it comprises at least four pneumococcal polysaccharide antigens of different serotypes. 8. An immunogenic composition according to any of claims 1 to 7, characterized in that the adjuvant comprises at least one of the following: 3D-MPL, a saponin immunostimulator, or an immunostimulatory CpG oligonucleotide. 9. An immunogenic composition according to claim 8, characterized in that the adjuvant comprises a vehicle selected from the group comprising: an oil-in-water emulsion, liposomes and an aluminum salt. 10. An immunogenic composition according to any of claims 1-9, characterized in that it is used as a medicament. 1 1. A vaccine characterized in that it comprises the immunogenic composition according to claims 1-9. 12. A method for the prevention or reduction of infection by Streptococcus pneumoniae in a patient over 55 years of age, characterized in that it comprises the administration of an effective amount of a vaccine comprising a polysaccharide of Streptococcus pneumoniae, at least one Streptococcus protein pneumoniae and a TH 1 -inducer adjuvant. 13. The use of a pneumococal polysaccharide antigen in combination with a protein antigen of Streptococcus pneumoniae and a TH 1 -inducer adjuvant, in the development of a drug for the prevention of pneumonia in patients over 55 years. A method for the preparation of the immunogenic composition according to claims 1-10, characterized in that it comprises the steps of: selecting one or more pneumococcal polysaccharide antigens; select one or more pneumococcal protein antigens; selecting a TH1-inducing adjuvant; And mix said polysaccharide and protein antigens and the adjuvant with a suitable excipient. 5. A method for the prevention or reduction of otitis media in infants, characterized in that it comprises the administration of a safe and effective amount of a vaccine comprising a polysaccharide antigen of Streptococcus pneumoniae, a protein antigen of Streptococcus pneumoniae and an adjuvant inductor of TH 1, to said infant.