MXPA00006324A - Adjuvanted vaccine formulation - Google Patents

Abstract

The present invention provides vaccine formulations comprising T-independent or polysaccharide conjugate vaccines adjuvanted with an immunostimulatory CpG oligonucleotide.

Description

VACCINE FORMULATION WITH AUXILIARY DESCRIPTION OF THE INVENTION The present invention relates to new vaccine formulations, and to methods for their production and their use in medicine. Immunomodulatory oligonucleotides containing non-methylated CpG dinucleotides ("CpG") are known (WO 96/02555, EP 468520). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. Historically, it has been observed that the BCG DNA fraction can exert an antitumor effect. In other studies, it was shown that synthetic oligonucleotides derived from the BCG gene sequences are capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a CG central motif, carried this activity (Tokunaga, T. et al., Microbial, Immunol.36: 55 (1992).) The central role of the CG motif in the immunostimulation afterwards was shown in a publication by Krieg (Nature 374 p546 1995) Detailed analysis has shown that the CG motif has sequences that are common in bacterial DNA but are rare in vertebrate DNA.It is currently believed that this difference in evolution allows the system The immune response of vertebrates detects the response of bacterial DNA (as if it occurred during an infection) leading consequently to the stimulation of the immune system.The immunostimulant activity has been shown for sequences as small as 15 nucleotide bases (Krieg, et al., Nature 374: 546 (1995)) and that the CpG motif has to be non-methylated.It has been postulated that the oligo must be a hexamer binding: purine, purine CG p irimidine pyrimidine, but this is not mandatory. Streptococcus pneumoniae is a gram positive bacterium that is pathogenic to humans, causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute otitis media. The mechanisms through which the pneumococcus extends to the lungs, brain spinal fluid and blood are poorly understood. The growth of bacteria that reach the normal pulmonary alveoli is inhibited by their relative dryness and by the phagocytic activity of alveolar macrophages. Any anatomical or physiological derangement of these coordinated defenses tends to increase the susceptibility of the lungs to infection. The cell wall of Streptococcus has an important role in generating an inflammatory response in the alveoli of the lung (Gillespie et al., I &65: 3936). The release of cell wall components occurs at the end of the pneumococcal development cycle through autolysis due to the synthesis of the N-acetyl-muramoyl-L-alanine-amidase (lytA) protein. The DNA will also be released into the infected region after pneumococcal autolysis. In order for the body to have an effective immune response against the invading bacteria, it must have mechanisms to coordinate the type of immune response most likely to stop the infection. For intracellular pathogens, coordination seems to occur between immune responses mediated by cell or humoral, and these are controlled by Th1 and Th2 type T cells. However, extracellular bacteria often employ a polysaccharide either in the form of a capsule or a lipopolysaccharide to protect them from the effects of serum complement, which can lyse bacteria, or make them accessible to phagocytes such as macrophages and neutrophils. . In this case, the immune response follows another trajectory, the immune response independent of T. The immune response independent of T can also be divided into type 1 and type 2. Type 2 antigens independent of T possess the modality characteristics by antigens of polysaccharides, including two weights: large molecular weight, repeat antigenic epitopes, ability to activate the complement cascade, poor in vivo degradability and inability to stimulate the MHC class II dependent helper cell (Mond et al., Annu Rev Immunol 13: 655-92). Type 1 antigens, unlike polysaccharides, are mitogenic for B cells, and are composed of lipopolysaccharides (LPS). Type 2 antigens independent of T can not stimulate responses in neonatal mice or CBA / N mice carrying an immune B cell defect linked to X (xid mice), whereas type 1 antigens can. Type 2 antigens induce weaker antibody responses as compared to T-dependent antigens such as proteins. The proteins are able to activate B cells and induce the secretion of the antibody being processed to peptides and presented on the surface of the B cell in the context of MHC class II, allowing the B cell to interact with the T cells and receive signals additionally required for maximum B-cell proliferation and maturation. However, although oligosaccharides may, in certain cases, be associated with MHC class II (Ishioka et al., J. Immunol., 148: 2446-2451) and lipidated polysaccharides appear associating with CD1 present in lymphocytes, (Fairhurst, RM et al., Immunology Today 19: 257 (1998)), there is no known mechanism of presentation for type II antigens to T cells. However, the multiple nature of repeat polysaccharide polymer antigen can cause the interlacing of receptors on the surface of the B cell, leading to the activation of the B cell through a mechanism that does not require T cells. In this way, the polysaccharides are T-independent antigens and are characterized in animals and babies by the production of IgM antibodies and the lack of booster and immunological memory. Only adult humans can produce significant amounts of the IgG antibody to most (if not all) polysaccharide antigens. The ability to switch the isotope of antibody to IgG coincides with the appearance of complement receptor 2 (CR2) in the B cells of infants or children who are just beginning to walk between the ages of 1.5 to 2 years, and this may provide the signal additional required for the activation and maturation of B cells. The present invention, in one aspect, provides a vaccine formulation, which is capable of raising an immune response to the independent antigen T. The production of IgG antibodies to the capsular polysaccharides of bacteria it is essential since the main mechanism of protection against these bacteria, complement-mediated lysis and opsonophagocytosis, are very effective with this antibody isotope (Maslanka et al., Clin Diag Lab Immunol., 4: 156-67, and Romero-Steiner et al. , Clin Diag Lab Immunol 4: 415-22). Polysaccharide antigen-based vaccines are well known in the art, and four that have been licensed for human use include the Vi polysaccharide of Salmonella typhi, the PRP polysaccharide of Haemophilus influenzae, the tetravalent meningococcal vaccine composed of serotypes, A, C , W135 and Y, and the 23-valent pneumococcal vaccine composed of the polysaccharides corresponding to serotypes 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B , 17F, 18C, 19A, 19F, 20, 22F.23F, and 33. The last t vaccines provide protection against bacteria that cause respiratory infections that result in severe morbidity and mortality in babies, even these vaccines have not been authorized for used in children under two years of age, as they are poorly immunogenic in this age group. The licensed polysaccharide vaccines listed above have a different demonstrated clinical efficacy. The Vi polysaccharide vaccine has been estimated to be between 55% and 77% effective in preventing typhoid fever confirmed by culture (Plotkin and Cam, Arch Intern Med 155: 2293-99). The meningococcal polysaccharide C vaccine was shown to have an efficacy of 79% under epidemic conditions (De Wals P, and others, Bull World Health Organ. 74: 407-411). It was shown that the 23-valent pneumococcal vaccine has a wide variation in clinical efficacy, from 0% to 81% (Fedson et al., Arch Intern Med. 154: 2531-2535). The efficacy appears to be related to the risk group being immunized, such as elderly people, Hodkin disease, splenectomy, sickle cell disease and agammaglobulinemics (Fine et al., Arch Intern Med. 154: 2666-2677), and also to the manifestation of disease. Pneumococcal pneumonia and otitis media are diseases that have not shown protection through the 23-valent vaccine. It is generally accepted that the protective efficacy of the pneumococcal vaccine is more or less related to the concentration of antibody induced by vaccination; in fact, the 23 polysaccharides were accepted to be authorized only by the immunogenicity of each component polysaccharide (De Williams and others, New York Academy of Sciences 1995 pp. 241-249). To increase the antibody response to the pneumococcal polysaccharides comprising the 23-valent vaccine, the present inventors sought to improve the immune response through the addition of the immunostimulant QS21 EP 362 279 and dQS21 WO 96/33739; however, no increase in antibody responses to polysaccharides in Rhesus monkeys could be measured. Threadgill et al., Vaccine 1998 Vol 16 (1) p76 have recently reported that immunostimulatory CpG oligonucleotides reduce the polysaccharide-specific antibody response when the oligonucleotide is formulated with the polysaccharide Pseudomas aeruginosa. Surprisingly, the present inventors have found that it is possible to help the immune response to pneumococcal polysaccharide vaccines by formulating with an immunostimulatory CpG immunoprotein, said formulations providing an immune response that produces significant levels of IgG antibodies.

In accordance with the present invention, a vaccine composition comprising a polysaccharide antigen aided by an immunostimulatory oligonucleotide is provided. The polysaccharide antigen can be unconjugated or conjugated to a carrier protein in a manner that provides T helper epitopes. The oligonucleotides can be DNA or RNA, but preferably contain a hexamer motif: purine, purine, CpG pyrimidine, pyrimidine. Most preferably, the linkage between nucleotides is modified to increase the stability of the oligonucleotide. Preferred modifications are phosphorothioate linkages. The lytA protein involved in the catalytic degradation of the pneumococcal cell wall is produced at the time of autolysis, and is part of the competition-induced operon (Mol Microbiol 29: 1125 (1998)). By definition, the mRNA encoding lytA will be present in large quantities during the synthesis of the lytA protein. In addition, the lytA protein contains a binding region of fofsphoryl choline that contains DNA repeat sequences (Yother and Briles J Bacteriol, 174: 601 (1992)), and which can be found in many other choline binding proteins present. in Streptococci. The following CpG sequences were identified from phosphoryl choline binding regions of lytA and from choline binding protein A (cbpA) (Rosenow et al., Mol.Microbiol 25: 819-829 (1997)). OLIGO 1: GCTACTGGTACG TACATTC AGACGGC TCTT (lytA) OLIGO 2: ACTATCTAAACGCTAATGGTGCTATGGCGACAGGATGGCT (cbpA) and can be used in the present invention. The following sequences of oligonucleotide immunostimulants also form preferred embodiments of the invention. OILGO 3: TCC ATG ACG TTC CTG ACG TT OLIGO 4: TCT CCC AGC GTG CGC CAT The CpG and flanking sequences have been underlined, and there are conserved motifs ACGT, ACG and ACG. Sequences derived from the choline binding regions of pneumococcal proteins have two CpG motifs that repeated 10 or 15 separate nucleotide bases, and a motif based on this nucleotide base distance between two CpGs occurs three times and five times, respectively, in the lytA and CbpA proteins. However, the published sequences have two CpG motifs that are seven or two separate nucleotide bases. In one embodiment, when combined with the commercially available 23-valent polysaccharide vaccine (Pneumovax, Pasteur Merieux), the auxiliary aspect of CpG significantly increased (IgG antibody) especially to the polysaccharide types 19F and 14 when administered intramuscularly. Thus, advantageously in one embodiment of the present invention, it is possible to improve the efficacy of a commercially available pneumococcal vaccine. This is particularly important in high-risk populations, especially those that have suboptimal antibody responses to polysaccharides. Such populations may include, but are not limited to, older people, patients with any of the following: splenectomy, congenital asplenia, hypoplegia, falsiform cell disease, cyclic neutropenia, drug-induced neutropenia, aplastic anemia, congenital agammaglobulinemia, hypogammaglobulinemia, deficiency of subclass selective IgG, multiple myeloma, chronic lymphocytic leukemia, lymphoma, HIV infection, conditions of multiple factors such as treatment with glucocorticoids, poor nutrition, cirrhosis of the liver, renal failure, diabetes mellitus, alcoholism, chronic disease, hospitalization, fatigue, tension, exposure to cold, previous respiratory infection, influenza and asthma. It may also include healthy adults such as healthy workers, military, prisoners or others, including travelers and those who go to schools who want to ensure complete vaccine coverage. In a preferred application, the CpG adjuvant is used to increase the response to the polysaccharide vaccine when it is used as a booster in children aged 6 and 24 months who have received their first immunization with a multivalent polysaccharide-pneumococcal protein conjugate. Said vaccines used for primary immunization are also advantageously assisted with a CpG oligonucleotide. Therefore, in one embodiment, a method for immunizing a patient is provided, comprising administering an effective amount of a vaccine according to the invention. In a second embodiment, a method is provided for reinforcing an immune response to a subject previously initiated to an antigen by administering a T-independent antigen with an immunostimulatory oligonucleotide CpG. The adjuvant treatment of CpG can be applied, according to the present invention, to other vaccines based on polysaccharide antigen and T-independent. These include, but are not limited to, Vi polysaccharide vaccine against Salmonella typhi, the polysaccharide vaccine tetravalent meningococcal (comprising types A, C, W135 and Y), the modified polysaccharide and polysaccharides of meningococcus group B, polysaccharides of Staphylococcus aureus, polysaccharides of Streptococcus agalactae, polysaccharides of Mycobacteria, for example, Mycobacterium tuberculosis, such as trehalose of manofasfoinisitides, mycolic acid, arabinomannans blocked at the end with mannose, the capsule of these and arabinogalactans, polysaccharides of Cryptococcus neoformans, the lipopolysaccharides of Haemophilus influenzae without type, the lipopolysaccharides of Moraxella catharralis, the lipopolysaccharides of Shigella sonnei, the lipopeptidofosfoglicanos (LPPG) of Trypanosoma cruzi, the g angliosides associated with cancer GD3, GD2, associated with tumor, especially the T-F antigen, and the sialyl T-F antigen, and the polysaccharide associated with HIV that is structurally related to the T-F antigen. Other T-independent antigens can be derivatives of Salmonella, cholera, Escherichia, Chlamydia and independent antigens of Plasmodium T. The vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.6! Vaccine Design- the subunit and adjuvant approach, edited by Powell and Newman, Plenurn Press, 1995. Encapsulation by liposomes is described by, for example, Fuller's patent of U.A. 4,235,877. The conjugation of proteins to macromolecules is described, for example, by Likhite patent of U.A. 4,372,945 and by Armor et al., Patent of E.U.A. 4,474,757. The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without adverse side effects, important in typical vaccines. This amount will vary depending on what specific immunogen is used and how it is presented. Generally, each dose is expected to comprise 0.1-1000 μg of the polysaccharide or polysaccharide-protein conjugate, preferably 2-100 μg, most preferably 4-40 μg. An optimal amount for a particular vaccine can be determined through standard studies that involve observation of appropriate immune responses in subjects. After an initial vaccination, subjects may receive one or more adequately separated booster immunizations. The oligonucleotides used in the present invention are typically deoxynucleotides. In a preferred embodiment, between nucleotides in the oligonucleotide is phophorodithithioate, or most preferably the phosphorothioate linkage, although phosphodiesters are within the scope of the present invention, other linkages between nucleotides that stabilize the oligonucleotide can be used. The CpG oligonucleotides used in the present invention can be synthesized through any method known in the art (for example EP 468 520) conveniently, such as oligonucleotides that can be synthesized using an automated synthesizer. Methods for producing phosphorothioate or phosphorodithioate oligonucleotides are described in the U.S. Patents. 5,663,153, E.U.A. 5,278,302 and WO 95/26204.

Example 1. Auxiliary of CpG of 23-valent pneumococcal polysaccharide in mice. The protection against pneumococcal infection was mediated through the IgG antibody to the capsular polysaccharide, together with the deposition of complement that makes the bacteria susceptible to annihilation by neutrophils through opsonophagocytosis. In this way, the protective efficacy of the vaccine can be estimated only based on the induction of IgG antibody. Groups of 10 mice were immunized once with a commercial 23-valent pneumococcal polysaccharide vaccine at human doses of 1/10, 1/50, or 1/250 (total polysaccharide 57.7.11.5 and 2.3 μg, respectively), and with CpG (50 μg of oligo 1), CpG + Alum. After immunization, serum concentrations of IgG to the 4 most important serotype polysaccharides (6B, 14.19F and 23F) were measured through ELISA every 7 days for 4 weeks.

MATERIALS AND METHODS The following groups were immunized. (10 balb / c mice per group): 23 Valente at 2.3,11.5 and 57.5 μg / dose (1 / 250,1 / 50 and 1/10 human dose). 23 Valente + CpG (50μg) in the same dose range. 23 Valente + CpG + AI (OH) 3 in the same dose range.

USED COMPONENTS FORMULATION PROCESS Pneumovax was diluted in H2O and concentrated 10mM PO4 ten times, 150 mM NaCl, pH 6.8, to obtain 2.3, 11.5 or 57.7 μg of antigen per dose. CpG was added for 30 minutes and for groups containing Al (OH) 3, the formulations were absorbed for 30 minutes both in AI (OH) 3 (50μg). Thiomersal (50μg / ml) was added as a preservative.

ELISA There were 10 animals per group, but since the bleeds were performed every week, only 5 animals per week were bled. ELISA and opsonophagocytosis assays were performed on poured sera. The ELISA assay was performed to measure murine IgG using the protocol derived from WHO Workshop in the ELISA procedure for the quantification of IgG antibody against capsular polysaccharides Streptococcus pneumoniae in human serum. In essence, the purified capsular polysaccharide was placed as a coating directly on the microtiter plate. The serum samples were pre-incubated with the cell wall polysaccharide common to all pneumococci and that is present in approximately 0.5% in purified pneumococcal polysaccharides according to the description (EP72513 Bl). Reagents from Jackson Immuno Laboratories Inc. were used to detect bound murine IgG. The titration curves were referenced to internal standards (monoclonal antibodies) modeled by the logistic equation. The calculations were made using SoftMax Pro software. The maximum absolute error in these results was expected to be within a factor of 2. The relative error is less than 30%.

RESULTS IgG isotope antibodies were found against serotypes 14 and 19G, but not against 6B and 23F, and the results for serotype 14 are presented in Figure 1. The response was dose dependent with 1/10 dose of human giving the highest response, indicating that the IgG response was specific for the polysaccharide. This is unusual, since mice normally only produced IgM against pneumococcal polysaccharides. The peak response was on day 14 after immunization, which is not unusual since the T-independent antigens do not induce memory. Individual analyzes were performed to determine the variation and statistical significance (data not shown). The response to the 23-valent human dose 1/10 was significantly (statistically) increased when assisted only with CpG (for type 19, GMC 0.8 compared to 3.7 μg / ml p = 0.07, type 14, GMC 0.19 compared to 3.4 μg / ml, p = .001). This was also true for 1/50 and 1/250 doses when measured with type 14. In addition, the responses were significantly increased for type 14 when assisted with CpG + Alum. The highest response was induced when the vaccine was only helped with CpG.

Example 2 Effect of the CpG auxiliary on the immunogenicity of conjugates PS-PD tetravalent pneumococcal in infant rat model.

The infant rat model was selected as published data showed that the relative immunogenicity of 4 pneumococcal polysaccharide protein conjugates in human babies was more similar to rats than mice. That is, 6B < 23F < 14 < 19F for baby rats. Baby rats were selected because their immune system may have developmental immaturity similar to that found in babies. The baby rats were immunized with a clinical grade batch of tetravalent pneumococcal polysaccharide (PD) conjugates in a 5-fold dose range and with the CpG and AIPO4 + CpG auxiliaries. We used I hear 1 at a dose of 100 μg. The animals were first immunized when they were 7 days old and received subsequent immunizations 14 and 28 days later. Serology was performed on samples from day 42 14 days after lll) and 56 (28 days after III). The best auxiliary was CpG alone: it increased the average concentrations of geometric IgG and the opsonic titers of 6B, 23F and 19F, while the titrations for serotype 14 were comparable with those preparations with auxiliary. The CpG-only formulation was also able to significantly increase seroconversion rates to serotype 6B-PD.

Materials and Methods Vaccine Groups The DSP0401x vaccine package contains the clinical grade lots of tetravalent PS-PD D6BPJ208 + D14PJ202 + D19PJ206 + D23PDJ212. ESPL001 contains the batches PS-LPD tetravalent E6BL040P + E14L66P + E19FL033P + E23FL21P.

Components used Formulation process Without tetravalent adsorbates The four conjugates were diluted in H2O and 10 times in 150mM of concentrated NaCl. As a concentrator, phenoxyethanol (500 μg / ml) was added. If CpG is necessary, the oligonucleotide is added to the non-adsorbed tetravalent. The isotonic character and the dissolution when necessary were ensured through NaCl.

Tetravalent adsorbed. The four monovalent adsorbed concentrates were diluted in H2O and 10 times in 150 mM concentrated NaCl before the addition of the A1PO4 component. Phenoxyethanol (500 μg / ml) was added as preservative.

If the dilutions are necessary, the tetravalent ones were diluted in A1PO4 to 1 mg / ml. These diluents were prepared in 150mM NaCl. If CpG is necessary, the oligonucleotide was added to the tetravalent adsorbed. The isotonic character was ensured through the addition of 1500 in mM of NaCl and if dilutions were required, diluents of A1PO4 were added at 1.3 or 1.8 mg / ml in NaCl. All formulations were prepared in glass jars without silicone.

Immunization protocol Baby rats were randomized to different mothers and were 7 days old when they received the first immunization. They received subsequent immunizations 14 and 28 days later. The bleeds were performed on day 42 (14 days after lll) and 56 (28 days after lll). All vaccines were injected s.c., and there were 10 rats per vaccine group.

ELISA ELISA was performed to measure rat IgG using protocol derived from WHO Workshop in the "ELISA procedure for the quantification of IgG against capsular polysaccharides of streptococcal pneumonia in human serum". In essence, the purified capsular polysaccharide was placed as a coating directly on the microtiter plate. The serum samples were pre-incubated with the cell wall polysaccharide common to all pneumococci and that was present in approximately 0.5% in purified pneumococcal polysaccharides. Jackson ImmunoLaboratories Inc. reagents were used to detect bound rat IgG. The titration curves were referenced to the titration curve of a reference serum molded through the logistic equation. The calculations were made using SoftMax Pro software. The standard sera were calibrated using a corollary response method, and the values were shown to correspond to estimates of Ig concentrations found by immunoprecipitation (reference 21).

Opsonophagocytosis The opsonophagocytic assay was performed following the CDC protocol (opsonophagocytosis of streptococcal pneumonia using differentiated HL60 cells, version 1.1). The modification included the use of domestic pneumococcal strains, and the phagocytic HL60 cells were replaced by purified human PMN. Rat polyclonal sera were included as a positive control.

Results Figure 2 shows the geometric mean concentrations of IgG produced against serotype 6B through the tetravalent combinations described in the materials and methods.

For clarity, the axes were divided between auxiliary and dose. Similar results were obtained against serotypes 19F and 23F, but type 14 had a more uniform response to all auxiliaries and doses. The biological activity of the antisera poured from each auxiliary group and dose was measured through opsonophagocytosis. The opsonic activity in relation to the concentration of IgG will give an estimate of the functional activity of the antisera. The values, shown in Table 1, illustrate that all adjuvants induce the antibody that has approximately the same capacity to opsonize pneumococci. In this way, CpG aids the induction of the specific antibody, and increases the concentration of antibody correlated with increases in protective efficacy.

Conclusion • A1PO4 (compared to non-auxiliary) significantly increases the seroconversion rate, geometric mean concentration of IgG, opsonic activity and immunological memory to tetravalent PS-PD. • The dose of 0.1μg significantly is more immunogenic than the dose of 0.5μg for serotypes 6b, 19F and conjugates 23F PS-PD in A1PO4. • IgG concentrations increased significantly against serotypes 6B, 19F and 23F when the conjugate vaccine was aided with CpG compared to A1PO4. This was confirmed through increased seroconversion rates and high opsonophagocytic titers.

TABLE 1 Relative opsonic activity (IgG concentration required for 50% annihilation of pneumococci) compared by serotype and auxiliary Example 3 Effect of the CpG auxiliary on the immunogenicity of 11-valent pneumococcal PS-PD conjugates in the baby rat model.

Example 2 showed that the CpG auxiliary of conjugate vaccines resulted in several increments of the order of 5 to 10 times more than with conventional auxiliaries (aluminum). In order to determine if these effects were dependent on the oligo sequence, dose or formulation, other experiments were performed. CpG OLIGO 2 was selected and used at a lower dose, this was 1 to 10 μg. It was also adsorbed on AI (OH) 3, and combined with the conjugate vaccines. In addition, since the immunological characteristics of each polysaccharide may be different, 11 serotypes were tested.

Materials and methods TABLE 2 Selection of PS-PD pneumococcal lots Formulation To examine the effect of different advanced adjuvants, the dose of conjugate was kept constant at 0.1 μg of each polysaccharide, and the auxiliaries AIPO4, AI (OH) 3 and CpG were formulated in different doses and combinations. In total, 10 different combinations were tested, without including the auxiliary at all. These are listed numerically in Table 3 for reference.

Preparation of diluents Two diluents were prepared in 150mM NaCl / phenoxy A: AIP04 at 1 mg / ml. B: CpG in AI (OH) 3 at 200 and 1000 μg / ml respectively. Weight ratio CpG / AI (OH) 3 = 1/5 Preparation on 11-valent adsorbed The eleven monovalent PS-PDs adsorbed, concentrated were mixed at the correct ratio. The complement of AIPO4 was added. When necessary, CpG (CpG adsorbed on AI (OH) 3) or diluent was added. Preparation of 11-valent not adsorbed The eleven PS-PD conjugates were mixed and diluted to the correct ratio in 150 mM NaCl pH 6.1, phenoxy. When necessary, CpG was added either as a solution (not adsorbed) or as CpG adsorbed on AI (OH) 3. The formulations for all injections were prepared 18 days before the first administration.

TABLE 3 Summary table of 11-valent pneumococcal PS-PD-tested auxiliary formulations in baby rats Immunization protocol Baby OFA rats were randomized to different mothers with an age of 7 days 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 III). All vaccines were injected s.c., and there were 10 rats per vaccine group.

ELISA The ELISA assay was performed as described in Example 2.

Opsonophagocytosis The opsonophagocytic assay was performed following the CDC protocol (Opsonophagocytosis of streptococcal pneumonia using differentiated HL60 cells, version 1.1). The modification included the use of domestic pneumococcal strains, and the phagocytic HL60 cells were replaced by purified human PMN. In addition, 3mm glass beads were added to the microtitre cavities to increase mixing, and this allowed the reduction of the phagocyte: bacteria ratio, which was recommended to be 400.

Results Tables 4 to 7 below show the geometric mean IgG concentration, the seroconversion rate and the opsonophagocytic arithmetic mean titration determined for 4 pneumococcal serotypes after immunization with a pneumococcal PS-Protein D conjugate vaccine. 11-valent with different formulations of CpG OLIGO 2. Compared with no auxiliary 10 μg of CpG induced higher concentrations of IgG for all serotypes. CpG induced significantly higher IgG concentrations than AIPO4 for serotypes 1,6B, 18C and 19F. For comparison, the results of example 2 are included in the tables using OLIGO 2. No significant difference in the IgG responses induced by the two OLIGO sequences was observed when OLIGO 2 was used at 10 μg. However, OLIGO 2 at 1 μg did not show any immunostimulant effect evidenced in that the IgG concentrations induced are not significantly different from the group without CpG. The adsorption of OLIGO 2 on AI (OH) 3 reduces the immunostimulatory effect, and the induction of the antibody is not significantly different from AIPO4 as an adjuvant.

TABLE 4 Geometric mean concentration of IgG in serotype 6B, in seroconversion and average opsonic titration on day 28 after immunization lll of baby rats with 11-valent PS-PD using different auxiliaries (And comparison with tetravalent immunization, Example 2) TABLE 5 Geometric mean concentration of IgG in serotype 14, seroconversion, and average opsonic titration on day 28 after immunization III in infant rats with 11-valent PS-PD using different adjuvants (and comparison with tetravalent immunization Example 2) TABLE 6 Geometric mean concentration of IgG in serotype 19F, seroconversion, and average opsonic titration on day 28 after immunization III in 11-valent PS-PD infant rats using different adjuvants (and comparison with tetravalent immunization, Example 2) TABLE 7 Geometric mean concentration of IgG in serotype 23F, seroconversion, and mean opsonic titration on day 28 after immunization III in 11-valent PS-PD infant rats using different adjuvants (and comparison with tetravalent immunization, Example 2) Example 5 Influence of CpG on the reinforcement with polysaccharide after starting with polysaccharide-conjugate vaccines, and on initiation with polysaccharide. The above examples have demonstrated the ability of CpG to aid the immune response to T-independent antigens, and to T-independent antigens harvested to a protein carrier. This led to consideration of whether CpG can help a memory response produced by reinforcement with an antigen independent of T after initiation with the T-dependent antigen. To determine these effects, mice were initiated either with polysaccharide pneumococcus or with pneumococcal polysaccharide aided with CpG or pneumococcal polysaccharide of protein D conjugate.

Immunization Protocol Balb / c mice 6 to 8 weeks of age were immunized subcutaneously with the vaccine formulations described below. The dose was 1 μg per polysaccharide for both conjugated and unconjugated formulations. A blood test was performed 14 days after measuring the IgG concentrations. After 56 days, another blood test was performed, and then a booster vaccination was given, and a final blood test was performed 14 days later, ie 70 days after the first immunization.

Group Initiative or Reinforcement 1 Saline Conjugate 2 PS PS 3 PS / Cp PS 4 PS Conjugate 5 PS / Cp Conjugate 6 Conjugated PS 7 Conjugated PS / CpG 8 Conjugate Conjugate Components used Formulation process The preparation of 4 monovalent adsorbed, concentrated (PS-PD conjugates). The monovalent, adsorbed, concentrates were prepared according to the procedure described above in Example 2.

Preparation of tetravalent (conjugates of PS-PD). The four monovalent adsorbed concentrates were mixed at the correct ratio (1 μg of each valence / dose) and diluted in NaCl, pH 6.1. The complement of AIPO4 (10μg / dose) was added as a diluent at 1mg / ml in 150mM NaCl pH 6.1 containing 5mg / ml phenoxyethanol. Preparation of tetravalent non-adsorbed, unconjugated with or without CpG (free PS). The four free PS were mixed at the correct ratio (1 μg of each valence / dose) and diluted in NaCl, pH 6.1. When necessary, CpG (100 μg / dose) was added. 5 mg / ml phenoxyethanol was added as preservative. jj &The formulations for both injections were prepared 6 days before the first administration in glass jars without silicone.

Formulation Process Preparation of 4 monovalent adsorbed, concentrated (PS-PD conjugates). The monovalent adsorbed, concentrated according to the procedure described above was prepared.

Preparation of tetravalent (PS-PD conjugates) The four monovalent concentrates, adsorbed, were mixed at the correct ratio (1 μg of each valence / dose). The complement of AIPO4 (10 μg / dose) was added as a diluent at 1 mg / ml in 150mM NaCl pH 6.1, containing 5mg / ml phenoxyethanol.

Preparation of tetravalent, not adsorbed, not coniugated with or without CpG (free PS) The four free PS were mixed at the correct ratio (1 μg of each valence / dose) and diluted in NaCl pH 6.1. When necessary, CpG was added. 5 mg / ml phenoxyethanol was added as preservative. The formulations for both injections were prepared 6 days before the first administration in glass jars without silicone.

ELISA The ELISA assay was performed as described in Example 1.

Results The results of this experiment are initiation and reinforcement. The results of the initiation with previous observations (Example 1) since an increased seroconversion and higher concentrations of IgG were found in mice that were immunized with polysaccharide aided with CpG compared to full polysaccharide. As found in Example 1, increases in the concentration of type 14 IgG with CpG helper are statistically significant compared to PS alone, and increases for the important type 19F appearance. However, the IgG concentrations with the CpG helper were not as high as was observed in example 1. To explain this difference, only two differences were made in the experiments, the valence of the vaccine (worth 23 versus 4 valent) and the route of administration (intramuscular versus subcutaneous) since the valence reduction is not expected to reduce immunogenicity, evidence indicates that the route of administration is important for an optimal CpG helper for T-independent antigens. This is consistent with a recent publication a failed attempt to use the CpG auxiliary for a full polysaccharide vaccine. The administration route used was interperitoneal (Threadgill et al., Vaccine 1998 Vol 16 (1) p (76). * p = 0.001 Fisher's extract test dp = 0.11 Fisher's extract test? p = 0.17 Fisher's extract test tjp = < 0.001 Fisher's extract test In the second part of this experiment, animals initiated either with PS, PS / CpG or conjugate vaccine were reinforced with PS, or with PS / CpG or with conjugates. To normalize the comparison data, the increase in IgG times was determined 14 days after giving the boost and the animal number showing an increase in antibody concentration was counted as the responder. * p = 0.09 Student's t test? p = 0.12 Student's t test dp = 0.03 Fisher's extract test Discussion This example confirms the results presented in Example 1, but has revealed that the mode of immunization may be important for optimal immunity. In an expression of the experiment for reinforcement and memory, two interesting features of the CpG helper were demonstrated. The first is that initiation with PS assisted by CpG leads to an increase of times greater after reinforcement with polysaccharide, and there is a tendency towards statistical significance. This could indicate that CpG was able to induce a better memory. The second feature is that CpG can aid a memory response induced by polysaccharide in animals initiated with conjugate vaccine.

Conclusions CpG is able to induce in mice an isotype switch of antibody against unconjugated polysaccharides. The magnitude of the IgG response is greater with CpG.

Claims (8)

1. - A formulation comprising an immunostimulatory CpG oligonucleotide and a polysaccharide antigen derived from Streptococcus Pneumoniae.

2. A formulation according to claim 1 wherein the polysaccharide is conjugated to a carrier protein.

3. A formulation according to claim 1 or 2, wherein the immunostimulatory CpG oligonucleotide has at least one internucleotide linkage, selected from phosphodiesters, phosphorodithioate and phosphorothioate.

4. A formulation containing a CpG oligonucleotide sequence according to any of claims 1, 2 or 3, which contains two CpG sequences that are separated by seven or more nucleotide base pairs.

5. A formulation according to claim 4, which contains two CpG sequences that are separated by 10 to 15 base pairs of nucleotide.

6. A formulation according to any of the preceding claims, wherein the CpG oligonucleotide is selected from the group: GCTACTGGTACG TACATTC AGACGGC TCTT ACTATCTAAACGCTAATGGTGCTATGGCGACAGGATGGCT TCC ATG ACG TTC CTG ACG TT TCT CCC AGC GTG CGC CAT 7.- A vaccine composition according to any of the preceding claims for use in medicine. 8. A method for inducing an immune response to a polysaccharide derived from the Streptococcus Pneumoniae antigen, said method comprising administering a safe and effective amount of a formulation as claimed herein, to a patient.