KR20010040898A - Pneumococcal and meningococcal vaccines formulated with interleukin-12 - Google Patents

Abstract

The present invention relates to a vaccine composition comprising an antigen such as pneumococcus or meningococcal antigen and a mixture of interleukin IL-12 that can be adsorbed on the mineral in suspension. The pneumococcal or meningococcal antigen may be conjugated to a carrier molecule. These vaccine compositions control the defense immune response to the antigen.

Description

Pneumococcal and meningococcal vaccines formulated with interleukin-12 {Pneumococcal and meningococcal vaccines formulated with interleukin-12}

A recent paradigm for the role of helper T cells in the immune response is that they can be separated into subgroups depending on the structure of the cytokines they produce, and the distinct cytokine profiles found in these cells determine their function. These T cell models include two major subgroups: TH-1 cells that produce IL-2 and interferon gamma (IFN-y) that increase both cellular and humoral immune responses, and increased humoral immune responses TH-2 cells (Mosmann et al., J. Immunol. 126: 2348 (1986)) that produce IL-4, IL-5 and IL-10. It is often desirable to improve the immunogenicity of the antigen in order to obtain a stronger immune response in the immunized organism and to enhance host resistance to the antigen-retaining agent. Substrates that enhance the immunogenicity of the antigen administered with the antigen are known as adjuvants. For example, certain lymphokines have been found to have adjuvant activity and to enhance the immune response to the antigen (Nencioni et al., J. Immunol. 139: 800-804 (1987): Howard et al., EP 285441).

SUMMARY OF THE INVENTION

The present invention relates to a vaccine composition comprising one or more pneumococcal or meningococcal antigens, interleukin IL-12 and a mixture of minerals in suspension. IL-12 can be adsorbed on the mineral suspension or simply mixed with it. In certain embodiments of the invention, IL-12 is adsorbed onto a mineral suspension, such as alum (e.g., aluminum hydroxide or aluminum phosphate). Such a vaccine composition modulates the protective immune response to the antigen, i.e., the vaccine composition can be used to quantitatively and qualitatively improve the antibody response of the vaccinated host, quantitatively increase cell-mediated immunity for defense responses to the pathogen . In certain embodiments of the invention, the antigen is a pneumococcal or a meningococcal antigen: the antigen is optionally conjugated to a carrier molecule, such as a pneumococcal or a meningococcal aqueous solution.

The studies described herein show that IL-12 is aluminum phosphate (AlPO ₄₎ as a dosage form pneumococci and can adjust the humoral response of mice immunized with meningococcal vaccines danggong liquid. The specific pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F (Pn1, Pn4, Pn5, Pn6B, Pn9V, Pn14, Pn18C, Pn19F, Pn23F) , And meningococcal polysaccharide is in C form (Men C). However, such serotypes are not intended to limit the scope of the invention, since other pneumococcal and meningococcal serotypes are also suitable for use herein. In addition, it will be clear to those skilled in the art that the conjugate with the carrier molecule, such as the CRM ₁₉₇ protein exemplified herein, is optional, depending on the immunogenicity of the selected S. pneumoniae or S. pneumoniae antigen.

IL-12 doses ranging from about 8 ng to about 1,000 ng increase IgG1, IgG2a, IgG2b and IgG3 responses to alum-adsorbed Pn14 or Pn6B. In addition, they increase the IgG2a response to Pn4 and Pn9V. An IL-12 dose of about 5,000 ng significantly reduced the total IgG titer to Pn14, particularly IgG1 and IgG2b titer.

The present invention also relates to a method of preparing an immunogen or vaccine composition comprising a mixture of an antigen and IL-12 together with a mineral in a suspension. In particular, IL-12 is adsorbed to mineral suspensions. The present invention also relates to a method of treating a defensive immune response comprising administering an effective amount of a vaccine composition comprising a mixture of an antigen, IL-12 and a mineral in a physiologically acceptable suspension solution to a mammal such as a human or primate host Lt; / RTI > producing T cells and a complement-binding IgG antibody of the vaccine vaccine. In particular, IL-12 adsorbs on mineral suspensions.

The immune system uses a number of mechanisms to attack pathogens; However, not all of these mechanisms are necessarily activated after immunization. Defense immunity induced by vaccination depends on the ability of the vaccine to induce an appropriate immune response to resist the pathogen or to remove the pathogen.

The studies described herein increase the immune response to alum-basic pneumococcal vaccines, particularly serotype 14 and serotype 6B pneumococcal conjugate liquid vaccines and meningococcal vaccines, particularly C forms, and the use of complement-binding IgG2a and IgG2b antibodies IL-12 < / RTI > As described herein, the PnPs-14-CRM ₁₉₇ vaccine comprises serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant of diphtheria toxoid (cross-reacting substance) represented by CRM ₁₉₇ , and PnPs6B-CRM ₁₉₇ The vaccine contains serotype 6B pneumococcal polysaccharide conjugated to CRM ₁₉₇ . IL-12 is compared to MPL ^R (3-O-deacylated monophosphoryl lipid A: RIBI ImmunoChem Research, Inc., Hamitton, Montana), a potent adjuvant for pneumococcal vaccine in mice. In individual experiments performed on Balb / c mice, the effect of IL-12 on the cytokine profile of CRM-specific T cells induced by the exemplified Allozyme conjugate vaccine was tested.

IL-12 is produced by various antigen-conferring cells, mainly macrophages and mononuclear cells. This is an important factor in the induction of TH-1 cells from simple T cells. The ability to produce or respond to IL-12 has been found to be important in the development of defensive TH-1-like responses, for example, during parasite infections, most notably in Reishmaniosis (Scott et al., US Pat. No. 5,571,515 number). The efficacy of IL-12 is mediated by IFN-y produced by NK cells and T helper cells. Interleukin-12 (IL-12), originally referred to as a natural killer cell promoter, is a heterodimer cytokine (Kobayashi et al., J. Exp. Med. 170: 827 (1989)). Expression and isolation of IL-12 protein in recombinant host cells is described in international patent application WO 90/05147 published May 17, 1990.

The studies described herein demonstrate the use of IL-12 as an adjuvant in pneumococcal or meningococcal vaccines, particularly pneumococcal or meningococcal liquid vaccines. Accordingly, the present invention relates to vaccine compositions comprising such antigens, IL-12 and mixtures of minerals in suspension. In certain embodiments of the invention, IL-12 is adsorbed onto a mineral suspension, such as alum (e.g., aluminum hydroxide or aluminum phosphate). Such a vaccine composition modulates a protective immune response to the antigen: the vaccine composition can induce a complement-binding antibody of the vaccinated host for a defense response against the pathogen. In a particular embodiment of the invention, the antigen is a pneumococcal antigen, in particular a pneumococcal polysaccharide: the pneumococcal antigen is optionally incorporated in a carrier molecule, such as a pneumococcal conjugate liquid. Although the specific pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F: since other serotypes are also suitable for use herein, Of the present invention.

In another embodiment of the invention, the antigen is a meningococcal antigen, in particular a meningococcal polysaccharide: the meningococcal antigen is optionally incorporated into a carrier molecule, such as a meningococcal synthetic fluid. C-type meningococcal bacteria have been exemplified herein; Since other forms are also suitable for use herein, this form is not intended to limit the scope of the invention.

IL-12 can be obtained from a number of suitable sources. This can be produced by recombinant DNA methodology, for example, by cloning a gene encoding human IL-12 and expressing it in a host system, while permitting the production of large amounts of pure human IL-12. Biologically active building blocks or fragments of IL-12 are also useful in the present invention. Commercial origins of recombinant human and murine IL-12 include the Genetics Institute, Inc. (Cambridge, Mass.).

The antigens of the present invention, such as pneumococcal or meningococcal antigens or pneumococcal or meningococcal aqueous fluids, can be used to induce an immune response against an antigen in a mammalian host. For example, the antigen may be serotype 14 or 6B pneumococcal polysaccharide or a portion thereof having an ability to promote an immune response. Additional suitable antigens include other encapsulated bacteria and their conjugates, secreted toxins, and polysaccharides from outer membrane proteins.

The method comprises administering an immunologically effective amount of a vaccine composition comprising a mixture of antigens, such as pneumococcal antigen or pneumococcal fluid, and an auxiliary amount of IL-12 adsorbed on the mineral in the suspension, to a mammal such as a human or primate .

As used herein, an " immunologically effective " dose of a vaccine composition is a dose sufficient to elicit an immune response. The specific dose of IL-12 and antigen will depend on the age, weight and medical condition of the mammal being treated and on the mode of administration. Appropriate doses will be readily determined by the technician. The vaccine composition may optionally be administered in a physiological saline solution or a pharmaceutically or physiologically acceptable vehicle such as an ethanol polyol such as glycerol or propylene glycol.

The vaccine composition may be a vegetable oil or an emulsifier thereof, a surfactant such as hexadecylamine, octadecyl amino acid ester, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dioctadecyl- N'bis (2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol and pluronic polyols; Polyamines such as pyran, dextran sulfate, poly IC, carbopole; Peptides such as muramyldipeptide, dimethylglycine, tosufin; Immune stimulating complex; Oil emulsifiers; MPL ^R < / RTI > and mineral gels. Antigens of the invention may also be incorporated into biodegradable polymers or ISCOMs (immunostimulatory complexes) such as liposomes, cochleates, poly-lactide, poly-glycolide and poly-lactide-co- Supplementary active ingredients may also be used. The antigens of the present invention may also be administered in association with bacterial toxins and their attenuated derivatives. Antigens of the present invention may also be administered in conjunction with other lymphokines, including, but not limited to, IL-2, IL-3, IL-15, IFN-y and GM-CSF.

Vaccines are not limited to humans or animals but can be administered in a variety of ways including, but not limited to, parenteral, intradermal, transdermal (by the use of slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, ≪ / RTI > The amount of antigen used in such a vaccine will vary depending on the nature of the antigen. Control and manipulation of a defined dosage range in which conventional carrier antigens are used for the vaccine modification of the present invention are well within the capability of those skilled in the art. The vaccine of the present invention is intended for use in the treatment of warm-blooded animals, especially humans, in both adulthood and adulthood. Typically, IL-12 and the antigen will be co-administered; In some instances, the skilled artisan will perceive that IL-12 may be administered at an approximate time before or after vaccination with the antigen.

The pneumococcal or meningococcal antigen of the present invention can bind to carrier molecules to regulate or enhance the immune response. Suitable carrier proteins include bacterial toxins that are safe and immunologically effective as a carrier by chemical or genetic methods of administration to mammals. Examples include non-toxic mutant proteins (cross-reactive substances (CRM)), such as pertussis, diphtheria and tetanus toxoid, and various non-toxic diphtheria toxoids, CRM ₁₉₇ . A simple toxin or fragment of toxoid containing at least one T-cell epitope is also useful as a carrier for the antigen and is an outer membrane protein complex. Methods for preparing conjugates of pneumococcal antigen and carrier molecules are well known in the art and are described, for example, in Dick and Burret, Contrib Microbiol Immunol. 10: 48-114 (Cruse JM, Lewis RE Jr, eds: Basel, Krager (1989)) and US Pat. No. 5,360,897 (Anderson et al.).

The adjuvant activity of IL-12 has a number of important implications. The assimilation of IL-12 may increase the concentration of protective antibody produced against the antigen in the vaccinated organism. The use of IL-12 as an adjuvant may improve the ability of the antigen to be weak or weakly immunogenic to induce an immune response. It can also be provided for safe vaccination if the antigen is toxic at the concentrations normally required for effective immunization. By reducing the amount of antigen, the likelihood of toxic reactions is reduced.

Typically, vaccination methods require the administration of the antigen over a period of several weeks or months to promote a " defensive " immune response. A defense immune response is an immune response sufficient to protect an immunized organism from a productive infection by a particular pathogen or a pathogen to the vaccine.

As shown in the embodiment, which typically comprises a serotype 14 or serotype 6B Streptococcus pneumoniae polysaccharide conjugated to inducing a reaction governed by a IGg1, CRM ₁₉₇ and the IL-12 adsorbed on AlPO _4, alum-formulated , 0.2 [mu] g of IL-12 substantially increased the IgG2 and IgG3 subclasses in both Balb / c and Swiss Webster mice, but had little or no effect on IgG1. An increase in IgG2b to Pn14 was found in Swiss Webster mice; 0.2 ㎍ of IL-12 has the same effect as the MPL gatneunde ^R 25 ㎍ the IgG subclass response to Pn14, in this connection, which will be described that IL-12 is at least 100 times more biologically active than the ^R MPL. IgG subclass distribution, in particular, as expected from the increased IgG2a response, opsonic inoculated activity of the antisera for the Pn14 Streptococcus pneumoniae from the received mouse awarded IL-12 0.2 ㎍ is formulated in a large lot large amounts of MPL ^R than that of the control group Lt; RTI ID = 0.0 > immunized < / RTI >

Briefly, IgG2a and IgG2b antibodies are highly effective in activating the complement system, while IgG1 antibodies are not. The complement system consists of a continuous plasma protein that travels around IGg2a or IgG2b bound to an antigen (e. G., Bacteria) to form a large molecular complex. The deposition of this complex on the surface of the bacteria can be accomplished by piercing the cell membrane (bactericidal activity), taking up the bacteria and killing them (opsonic bacteria), phagocytes (for example, polymorphonuclear cells (PMN) ), Which leads to bacterial death.

Increased IL-12 capacity significantly reduces IgG1 and IgG2b responses. The reduction of these immunoglobulin subclasses is due to changes in the kinetic of the antibody response simply as observed in the egg lysozyme (HEL) system (Buchanan, Van Cleave and Metzger, Abstract # 1945; 9th International Congress of Immunology , Since this subclass decreases at all time points tested. Switching of B cells to this subclass is expected to affect IgG1 if production requires IL-4, TH-2 cytokines that are inhibited by IL-12. However, a decrease in IgG2b is not expected in previous studies because increased levels of IgG2b correlate with the presence of TH-1-like T cells. It is likely that IFN-y, or IFN-y, as well as cytokines are involved in the regulation of IgG2b. For example, Germann et al. (Eur J. Immunol 25: 823-829 (1995)) found that mouse treatment with anti-IFN-gamma inhibited the ability of IL-12 to enhance IgG2a response, but not IgG2b. Other studies have included TGF-beta as an important factor in the induction of IgG2b (reviewed by J. Stavnezer, J. Immunol. 155: 1647-1651 (1995)). Without wishing to be bound by theory, high doses of IL-12 can affect TGF-beta production or its responsiveness.

IFN-y is important for the induction of IGg2a antibodies to T-dependent protein antigens (Finkelman and Holmes, Annu. Rev. Immunol. 8: 303-33 (1991)). IgG3 responds to T- , J. Exp. Med., 175: 1367-1371 (1992)). An enhanced IFN-γ response is consistently found on one days after vaccination and after the second immunization vaccine (PnPs-14-CRM ₁₉₇₎ containing IL-12 and AlPO _4. The effect of IL-12 on TH-2 cytokines IL-5 and IL-10 appears to be dependent on when lymphocytes are harvested after vaccination, and is also possible for certain cytokines. Exogenous IL-12 completely abolishes antigen-specific IL-5 and IL-10 production by harvested lymph node cells (LNC) one week after the first vaccination. After the second vaccination, there is a difference between these two cytokines; IL-5 production by LNC or splenocytes disappears completely with 1 μg of IL-12 in the vaccine, but IL-10 production is hardly affected after the second immunization. It is unclear whether this difference reflects the setting of the culture at different times or the expansion of the TH-2-like density with subsequent vaccination. The latter possibility is consistent with the data of Wolf et al. (Bliss et al., J. Immunol 156: 887-894 (1996)), suggesting that IL-4 producing T cells are preimmunized with a vaccine containing IL-12 Gt; Balb / c < / RTI > mice that were secondarily immunized with soluble antigens. In these studies, IL-4 is detected even when IL-12 is included in the second vaccine. The presence of TH-2 cytokines after secondary immunization can explain why in Balb / c mice, a much higher level of IL-12 can not reduce the secondary IgG1 response to lower control levels (Alumna conjugate vaccine ). Unlike Balb / c mice, the high dose of IL-12 severely inhibits the IgG1 response of Swiss Webster mice. It is clear that this is related to the reduced production of TH-2 cytokines after the second vaccination.

In this study, IL-12 acts as a "conventional" adjuvant and immunomodulator, depending on the vaccine, showing immunomodulatory activity. In studies with PnPs14-CRM ₁₉₇ , IgG responses to vaccines (especially primary responses) are not substantially improved by the presence of cytokines, but certain subclasses, IgG2a and IgG3, improve while others remain unchanged or decreased. Thus, IL-12 is already useful in regulating humoral responses to immunogenic vaccines. In this study, the adjuvant activity of IL-12 is likely to be interrupted by the presence of alum, an appropriate adjuvant on its own for highly immunogenic PnPs-14 conjugates. The assimilation of IL-12 may be better in the absence of alum, either by reducing the volume of the conjugate or by using a weakly immunogenic conjugate. Thus, a further evaluation is performed using IL-12 in the presence and absence of alum as a PnPs6B conjugated vaccine, which is less immunogenic than PnPs-4 conjugated vaccine in Swiss Webster mice.

Additional studies have been directed to address the problem of IL-12 adjuvant activity for weakly immunogenic pneumococcal conjugate liquids. Pn18C ball there is liquid is selected, which is the weak immunogenicity when formulated with AlPO _4, i.e., because the induced low titer IgG and all the mice do not react to this. When formulated as MPL or QS-21, more frequent reactions with high IgG titers can be achieved.

100 μg of MPL ^R and AlPO ₄ or 20 μg of QS-21 ^™ are the best adjuvants for the Pn18C reaction in this study because they induce the most frequent response to this serotype. Nevertheless, IL-12 significantly affects the IgG response to the carrier protein, CRM ₁₉₇ , in mice that were immunized with this conjugate. In addition, the effects of cytokines is regulated by the presence of the vaccine within the AlPO _4. Results in a dependence increasing - IL-12 action is certainly to, primary and secondary capacity of the IgG titers after vaccination as an adjuvant for the vaccine formulated without AlPO _4. IL-12 promotes IgG2a response to CRM ₁₉₇ , consistent with its ability to stimulate the induction of TH-1-like helper cells (IFN-y producer). However, IL-12 also enhances the IgG1 response to CRM ₁₉₇ after primary and secondary vaccination. IgG1 antibodies are typically associated with TH-2-like helper cells that produce IL-4.

The inclusion of 0.1 μg of IL-12 in the AlPO ₄ -modified Pn18C conjugate liquid vaccine, itself inducing a 10-fold increase in CRM ₁₉₇ response, does not affect IgG1 but substantially increases IgG2a titer. The thin IgG2a station achieved with IL-12 0.1 ㎍ as high as at least obtained with IL-12 5 ㎍ in the absence of AlPO _4. However, it should be noted that the presence of AlPO ₄ does not inhibit the enhancement of IgG1 response by IL-12. In mice immunized with AlPO ₄ phase Pn14 conjugate, a 0.2 μg dose of IL-12 increases the IgG1, IgG2a and IgG2b titers to CRM ₁₉₇ . Differences in the effect on IgG1 may reflect differences in the immunogenicity of the two conjugates for the CRM ₁₉₇ IgG response; The AlPO ₄ phase Pn14 conjugate liquid has space for IL-12 to induce a 10-fold lower CRM ₁₉₇ IgG titer and to enhance the IgG1 response, but not when the mice are immunized with AlnPO ₄ phase Pn18C conjugate. The fact that ^R MPL and QS-21 ^TM a significant increase in the IgG1 titer in mice immunized with AlPO ₄ phase Pn18C the conjugate shows that no IgG1 response is not promoted with up. Alternatively, the properties of the conjugated saccharide may be a factor. In both experiments, the high dose of IL-12 resulted in a significant reduction of the IgG1, IgG2a and IgG2b titers to CRM ₁₉₇ , which is not seen in the absence of AlPO ₄ .

IL-12 exerts its adjuvant effect to the extent that it is probably distinguishable from MPL ^R or QS-21 ^TM . IL-12 significantly increases CRM ₁₉₇ IgG2a titers in mice immunized with Pn18C conjugate, but has minimal effect on IgG2b. Conversely, MPL ^R and QS-21 ^TM increase the potency of both IgG subclasses. The separation of these two subclasses explains that IgG2b is not IFN-γ, which induces switching to IgG2a and mediates the immunomodulatory effect of IL-12, or is induced by cytokines in addition to IFN-γ. One candidate for inducing IgG2b production is TGFb. However, the nature of the antigen can not be ruled out because in mice immunized with Pn14 conjugated liquid, 0.2 [mu] g of IL-12 causes IgG2a and IgG2b to be increased to a similar level corresponding to increased potency by 25 [mu] g MPL ^R to be.

Capsules are R 2 consisting of the conjugate mixed with PnPs14-CRM ₁₉₇ conjugate of the polysaccharide using a vaccine from a serotype 6B Streptococcus pneumoniae (PnPs6B-CRM197) conjugated to CRM ₁₉₇ confirms the findings described above and extended . IL-12 not only regulates the IgG response to the Pn6B conjugate, but also increases the total IgG titer to the conjugate. Besides. The study is a relatively low dose adjuvant activity of IL-12 will be described further enhanced by this formulation to AlPO _4. Unlike the studies described above with PnPs-14-CRM ₁₉₇ synthetic fluid, IL-12 / AlPO ₄ increases both the IgG1 and IgG2a subclasses for Pn6B, suggesting that the Pn14 IgG1 response by IL- Explain that an apparent deficiency is probably not a phenomenon that can be created. This study further supports the discovery that the mechanisms of adjuvant activity by IL-12 and MPL ^R are not equivalent. Both adjuvants increase Pn6B IgG1 and IgG2a titer to similar levels, but MPL ^R is more effective at improving IgG2b and IgG3 antibodies.

IL-12 / AlPO ₄ does not act as adjuvants for Pn14 IgG response. The reason is uncertain; Not wanting to be included in the theory, this most reflects the fact that in previous studies, mice were immunized with 1 μg dose of PnPs-14-CRM ₁₉₇ aqueous solution, a 10-fold increase from the Pn6B study. The applicability of IL-12 to more complex pneumococcal vaccines is demonstrated using bimodal vaccines containing serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F of pneumococcal serotypes. The combination of IL-12 and AlPO ₄ , together with PnPs6B and PnPs14, increased the IgG2a antibody against PnPs4 and PnPs9V, and increased the concentration of an amphipathic liquid prepared from serotypes 18C pneumococcal saccharide (PnOs-18C-CRM ₁₉₇ ) ≪ / RTI > to the ability of the mouse to respond.

In a further embodiment, IL-12 is tested in an aqueous liquid vaccine against C-type meningococcal bacteria (MenC) and with an aqueous liquid vaccine against B-type Haemophilus influenzae (HbOC). Formulation of the vaccine with 50 ng IL-12 and AlPO ₄ increases the IgG2a titer against MenC capsular polysaccharides, but not HbOC.

The data presented herein demonstrate that AlPO ₄ can significantly enhance the potential of IL-12 to enable substantially lower amounts of cytokines to be used. One possible mechanism is that by binding to IL-12 AlPO _4, will improve the resistance thereof in an animal; Additional studies indicate that IL-12 binds rapidly to alum (data not shown). Alternatively, the local inflammatory effect of AlPO ₄ can lead to cytokines to enhance the biological activity of IL-12.

Along with an understanding of the physical interactions of IL-12 and AlPO ₄ , a number of other problems arise from this study with pneumococcal vaccines formulated with IL-12. If AlPO ₄ improves the activity of IL-12, it is necessary to determine the minimum amount of cytokine required to support the IgG response to the pneumococcal conjugate liquid and the minimum amount of cytokine required to stimulate IL-5-producing T cells by the IL- It will be useful to know if it is. These two questions are explained in the study of Balb / c mice described in Example 4.

The following examples are provided for the purpose of illustrating the invention and are not intended to limit the scope of the invention. The teachings of all references cited herein are incorporated herein by reference.

Example 1: Effect of IL-12 on the IgG response of Swiss Webster mice to serotype 14 pneumococcal capsular polysaccharide (PnPs-14-CRM / AlPO ₄ ) conjugated to CRM ₁₉₇ on aluminum phosphate

Research Design

Swiss Webster mice (10 per group) are immunized twice with 1 μg of PnPs-14-CRM ₁₉₇ formulated with 100 μg of AlPO ₄ and IL-12, or 0.2 μg, 1 μg or 5 μg of IL-12 Week and 3 weeks). All vaccines contain 0.25% normal mouse serum for the purpose of stabilizing IL-12 when used at low concentrations. PnPs14-CRM ₁₉₇ is a conjugate of capsular polysaccharides from serotype 14 pneumococci covalently linked to diphtheria toxin, CRM ₁₉₇ , genetically decoded by reductive amination. The other group receives 25 μg of MPL ^R (3-O-deacylated monophosphoryl lipid A, RIBI Immunochem Research, Inc., Hamilton Montana) instead of IL-12. Vaccination is carried out by subcutaneous route divided into 3 weeks. Serum is collected at 3 weeks (primary response) and at 5 and 7 weeks (secondary response at 2 and 4 weeks after secondary immunization). Serum is analyzed for IgG antibodies against PnPs-14.

Serum is also assayed for its ability to enhance opsoninogenesis of form-14 pneumococcal by human polymorphonuclear cells (PMN). 14 form pneumococcus is cleared with C8-deleted serum as the origin of the diluent and complement of the antiserum. These are then cultured in human polymomocyte-clear cells (PMN), and the% of surviving bacteria is determined by colony count.

result

Table 1 shows the IL-12 1 ㎍ Sikkim and 5 ㎍ This substantially reduces the anti -PnPs-14 IgG responses in mice immunized with the conjugate formulated in AlPO _4. The lowest dose of cytokine (0.2 μg) does not affect the total IgG response, but it causes major changes at the level of each immunoglobulin subclass. At 5 and 7 weeks (at 2 weeks and 4 weeks, respectively, after the second immunization), 0.2 [mu] g of IL-12 induces substantially higher titers of IgG2a, IgG2b and IgG3, but the level of IgG1 is essentially unchanged. Induced by IL-12 0.2 ㎍ IgG subclasses profile was not distinguished from those obtained with MPL ^R 25 ㎍, serum of awarded these adjuvants mouse is higher than that of mice immunized with a vaccine containing only AlPO ₄ And has opsonin phylum activity (Table 2).

Higher doses of IL-12 significantly reduce IgG1 antibodies; At 5 ug of cytokine, the IgG1 potency is at least 10 times lower than that immunized with IL-12. This effect is evident both during the first reaction and after the second immunization. Increases in IL-12 capacity do not further increase IgG2a, IgG2b and IgG3 and similar IgG1, but they also decrease to varying degrees. IgG2b showed the greatest reduction such that vaccines containing 1 [mu] g or 5 [mu] g IL-12 induced the same IgG2b titer as those without adjuvant. IgG2a and IgG3 have a high IL-12 dose; Are less sensitive to the effect of 5 [mu] g of IL-12, and these subclasses after the second vaccination are higher than in the control.

This study is the IL-12 can adjust the IgG subclass response to the conjugate vaccine, PnPs14-CRM ₁₉₇ formulation with AlPO _4. A dose of 0.2 [mu] g of IL-12 increases IgG2a, IgG2b and IgG3 responses to Pn14 without affecting the IgG1 response. Higher doses of IL-12 result in significant reductions in IgG1 and IgG2b titers. IgG2a and IgG3 transients also appear to decrease at this dose, but they are higher in the absence of IL-12 than in immunized mice. Example 2 indicates that IgG subclass changes are associated with IFN-y production, improved induction of CRM197-specific T cells, and significant reduction of antigen-specific IL-5 production, suggesting that the T helper cell phenotype is TH-2 - Describes the change from similar to TH-1-like.

Influence of IL-12 on the immunogenicity of PnPs-14-CRM ₁₉₇ / alum vaccine PnPs14 IgG reaction time Supplements Capacity (㎍) IgG IgG1 IgG2a IgG2b IgG3 3 weeks none 56,035 8,394 481 298 1,312 IL-12 5 13,137 480 2,417 388 2,398 IL-12 One 26,131 1,521 3,249 736 3,858 IL-12 0.2 90,220 13,779 4,731 1,454 7,944 MPL ^R 25 46,451 14,303 1,506 8,506 18,203 5 weeks none 531,270 189,571 5,507 14,463 18,158 IL-12 5 231,015 16,900 28,719 6,002 56,982 IL-12 One 198,044 36,327 27,420 11,841 30,740 IL-12 0.2 722,360 305,623 60,701 89,397 99,794 MPL ^R 25 751,066 221,324 44,957 91,265 77,989 7 weeks none 694,741 244, 212 1,801 6,849 9,245 IL-12 5 177,438 17,232 20,276 3,494 26,859 IL-12 One 183,571 44,213 21,246 5,063 13,447 IL-12 0.2 852,292 251,157 37,104 37,717 88,933 MPL ^R 25 783,622 187,055 30,694 89,153 59,297

Obsonin phylum activity of mouse serum immunized with PnPs-14-CRM ₁₉₇ / AlPO ₄ formulated with IL-12 or MPL ^R Bacterial survival rate% 5 week serum 7 week serum The initial serum dilution tested No IL-12 0.2 [mu] g IL-12 1 [mu] g IL-12 5 [mu] g IL-12 25 [mu] g MPL ^R No IL-12 0.2 [mu] g IL-12 1 [mu] g IL-12 5 [mu] g IL-12 25 [mu] g MPL ^R 2 6 10 6 6 9 6 6 7 7 5 8 12 7 9 9 7 21 4 10 9 6 16 32 4 24 25 3 47 8 17 26 8 32 71 12 61 94 23 68 6 85 90 21 64 64 46 90 89 51 116 34 79 99 76

Example 2: Characterization of T helper cells induced by pneumococcal conjugate liquid vaccine (PnPs-14-CRM ₁₉₇ / AlPO ₄ ) formulated with IL-12

Research Design

To a Balb / c mouse of eight groups in the tail base with AlPO ₄ 100 ㎍ and the PnPs-14-CRM ₁₉₇ conjugate formulated in 1 ㎍ IL-12 of different capacities immunized by subcutaneous route. Conventional mouse serum (0.25%) is included as a carrier protein. After one week, the efflux lymph node cell suspension is prepared from half of each group of mice and cultured for 6 days with CRM ₁₉₇ , lysozyme, Con A or in single medium. Culture supernatants from comparative cultures were harvested at 3 and 6 days and analyzed for IFN-y, IL-5 and IL-10 by ELISA.

At 3 weeks, the remaining mice are sampled and re-immunized with the same vaccine formulation used for the primary immunization. On the 14th day after the second immunization (5 weeks), the mice are once again bled. After 4 days, their draining lymph node cells and splenocytes are harvested and cultured for 6 days with CRM ₁₉₇ , lysozyme, ConA or in a single medium. Culture supernatants from comparative cultures were harvested at 3 and 6 days and analyzed for IFN-y, IL-5 and IL-10 by ELISA.

result

The PnPs-14-CRM ₁₉₇ / AlPO ₄ vaccine formulation formulated with low doses of IL-12 (0.2 μg and 1.0 μg) greatly enhanced IgG2a and IgG3 responses to Pn14 at 5 weeks, but not IgG1 ). Several previous studies have shown differences between the results obtained with Balb / c mice and Swiss Webster mice; In this experiment, IL-12 does not significantly increase the IgG2b antibody to Pn14, and 5 μg of IL-12 does not significantly (> 10-fold) decrease the IgG1 titer as compared to the control group without cytokines.

At 1 week after immunization, lymph node cells immunized with IL-12 produce IFN-y, IL-5 and IL-10 when promoted to CRM ₁₉₇ in vitro (Table 4). Addition of IL-12 to the vaccine significantly increases the antigen-specific production of IFN-y and eliminates the ability of lymphocytes to produce IL-5 and IL-10. Maximum IFN-y production was obtained with the lowest dose of IL-12 (0.2 [mu] g): high dose, especially 5 [mu] g, seems to decrease the level of this cytokine. This is most clearly seen in cultures promoted with 1 μg / ml CRM ₁₉₇ . Decreased IFN-γ at high doses of IL-12 can not reflect generalized compression because IFN-γ production in the response to Con A is identical regardless of the dose of IL-12 in the vaccine.

At two weeks after the second immunization, the lymph node cells and splenocytes immunized with the vaccine containing IL-12 showed an increased level of IFN-y in the CRM ₁₉₇ -promoted response compared to mice immunized without IL-12 . As observed at day 7 after the first vaccination, 0.2 ug to 1.0 ug of IL-12 is the optimal amount of IL-12 for the enhancement of the IFN-γ response. However, conversely, IL-5 and IL-10 production are affected differently. The 1.0 μg and 5.0 μg contents of IL-12 essentially reduced the IL-5 response, but by comparison had minimal effect on IL-10 production. IL-12 (5.0 [mu] g) abolishes the ability of splenocytes not IL-10 producing lymph node cells (Tables 5 and 6).

Effect of IL-12 on Immune Response to Alum-Basic PnPs14 Urinary Fluid Vaccine in Balb / c Mice week IL-12 capacity IgG IgG1 IgG2a IgG2b IgG3 3 none 41,480 7,347 1,387 895 2,333 0.2 26,253 1,521 1,118 171 5,911 One 26,124 966 2,155 248 5,991 5 10,753 541 671 183 3,242 5 none 234,220 33,284 2,896 3,105 2,487 0.2 674,996 71,808 18,245 6,789 107,470 One 632,714 32,022 22,749 7,853 44,350 5 224,832 19,495 10,083 1,287 25,212

Example 3: IL-12 adjuvant activity of weakly immunogenic pneumococcal conjugate liquid

Research Design

Swiss-Webster mice immunized with (10 animals per group) to Pn18C conjugate 1 ㎍ one in 100 ㎍ AlPO ₄ or formulated without AlPO _4. Vaccine is supplemented with IL-12 (0.2, 1 or 5 ㎍), 100 ㎍ MPL ^R or 20 ㎍ QS-21 ^TM. Conventional mouse serum (final 0.5%) is used to stabilize diluted IL-12 and added to all vaccines, regardless of composition. Three weeks later, mice are drawn and immunized secondary to the same vaccine formulation used in the primary immunization. Blood collection is also taken at 5 and 7 weeks of the study (2 and 4 weeks, respectively, after the second immunization). Pooling serum is tested for 5 weeks for Pn18C and CRM ₂₉₇ total IgG and IgG subclasses. To determine the frequency of response to Pn18C, serum from each mouse is diluted 1/500 and tested for IgG antibodies against Pn18C by ELISA. The results are reported as optical density.

result

The Pn18C IgG response is shown in Table 7. The addition of IL-12 to the alum-formulated conjugate vaccine does not have a consistent effect on the IgG response to Pn18C. A dose of 5 μg of IL-12 resulted in a 3-fold increase in the IgG titer of 5 full rounds of serum, whereas a vaccine formulated at 1 μg of IL-12 did not induce the Pn18C response. The lowest dose of IL-12 (0.1 μg) induces the same response as an AlPO ₄ -formulated vaccine that does not contain IL-12. The vaccine formulated with MPL ^R / AlPO ₄ induced a high frequency response: 7/10 mice achieved an OD> 0.2 compared to QS-21 ^TM / AlPO ₄ and AlPO ₄ alone, each of which received a 4/10 response . IL-12 and AlPO ₄ mice immunized with the vaccine containing induces respectively, 0.1 ㎍, 1.0 ㎍ and 2/10 in the IL-12 dose of 5 ㎍, 0/10 and 1/10 of the reaction.

In this experiment MPL ^R and QS-21 ^TM increase the Pn18C IgG response by at least 3 to 4 times. In the absence of AlPO _4, IL-12 does not have a clear adjuvant effect in Pn18C IgG response. Vaccines containing 1 μg dose of IL-12 induce the same Pn18C response as the vaccine without IL-12. Vaccines containing low and high doses of IL-12 appear to induce a lower response than the control vaccine. MPL ^R or QS-21 ^TM is not likely to improve the Pn18C IgG response. Of the vaccines formulated without AlPO ₄ , QS-21 ^™ induces the most frequent responders (7/10 with OD> 0.2), whereas all other formulations induce at most 4/10 responders.

To confirm that IL-12 in vaccines is actually active, the CRM ₁₉₇ IgG response is assessed in these mice. Tables 8 and 9 show that IL-12 increases the CRM ₁₉₇ IgG response dose-dependently in mice immunized with a vaccine formulated without AlPO ₄ after primary (3 weeks) and secondary (5 weeks) vaccination. In addition, there is an IL-12 dose-dependent increase in both IgG1 and IgG2a titers at 3 and 5 weeks, and IgG2b increases at 5 weeks. IgG1 and IgG2a transcripts at 5 weeks are similar to those induced by the vaccine formulated with 100 ug of MPL ^R. Conversely, the IgG2b potency enhanced by IL-12 is 20 times lower than that induced by MPL ^R. These data demonstrate that IgG2a and IgG2b are regulated by different mechanisms, IgG2a is dependent on the mechanism activated by IL-12, and IgG2b is regulated by an IL-12-independent mechanism. This data clearly demonstrates that IL-12 can serve as an adjuvant for IgG responses to protein antigens. In addition, an increase in IgG1 and IgG2a titers are Sikkim at least IL-12 improves the priming of both TH-1- like and TH-2- Similar helper cell by PnOs18C-CRM ₁₉₇ conjugate in the absence of AlPO ₄ in this model .

When 0.1 μg dose of IL-12 is added to the Pn18C conjugate vaccine formulated with AlPO ₄ , it increases slightly if the total IgG response to CRM ₁₉₇ is increased at 3 weeks, but increases 3-fold at 5 weeks. However, this dose of IL-12 increases the titer of IgG2a at 5 weeks and improves potency similar to that induced by vaccines containing MPL or QS-21. As shown in the experiments above, higher doses of IL-12 result in a slight decrease in IgG titers and all subclasses are affected.

Influence of IL-12 on CRM ₁₉₇ IgG response at 3 weeks after PnOs18C conjugate vaccination Supplement (㎍ / dose) IgG IgG1 IgG2a AlPO ₄ 70,964 8,706 3,516 0.1 ㎍ IL-12 + AlPO ₄ 103,589 4,754 13,025 1.0 ㎍ IL-12 + AlPO ₄ 26,927 506 2,926 5.0 ㎍ IL-12 + AlPO ₄ 19,579 241 2,665 100 ㎍ ^R MPL / AlPO ₄ 651,315 92,245 79,508 QS-21 ^TM / AlPO ₄ 572,255 116,583 38,419 none 7,630 452 1,023 0.1 [mu] g IL-12 32,403 3,475 3,713 1.0 [mu] g IL-12 60,987 4,615 5,951 5.0 [mu] g IL-12 128,697 10,498 10,686 100 [mu] g MPL ^R / TEM 462,289 40,010 24,979 QS-21 ^TM 556,440 111,533 53,799

Effect of IL-12 on CRM ₁₉₇ IgG response at 5 weeks after vaccination with PnOs18C conjugate (2 weeks after the second immunization) Supplement (㎍ / dose) IgG IgG1 IgG2a IgG2b AlPO ₄ 634,631 102,974 45,955 8,812 0.1 ㎍ IL-12 + AlPO ₄ 2,225,000 88,204 317,083 16,869 1.0 ㎍ IL-12 + AlPO ₄ 105,765 8.018 12,598 1,096 5.0 ㎍ IL-12 + AlPO ₄ 71,618 1,582 13,806 744 100 ㎍ ^R MPL / AlPO ₄ 4,384,000 637,655 371,652 111,646 QS-21 ^TM / AlPO ₄ > 5,000,000 > 1,000,000 873,674 144,132 none 62,341 12,783 3,655 1,679 0.1 [mu] g IL-12 296,791 52,288 23,741 7,069 1.0 [mu] g IL-12 1,026,060 101,381 96,024 11,862 5.0 [mu] g IL-12 1,367,771 74,494 108,815 14,258 100 [mu] g MPL ^R / TEM 4,173,765 264,691 266,160 303,662 QS-21 ^TM > 5,000,000 1,303,508 445,712 131,991

Example 4: Effect of IL-12 on the IgG response of Swiss Webster mice to a divalent vaccine containing PnPs6B-CRM ₁₉₇ and PnPs-14-CRM ₁₉₇

Research Design

Vaccine of the Swiss Webster mouse including 0.1 ㎍ and 0.1 ㎍ per dose of PnPs14-CRM ₁₉₇ danggong liquid per dose of PnPs6B-CRM ₁₉₇ danggong liquid (CRM ₁₉₇ and shared bound serotype 6B conjugate the capsular polysaccharide from Streptococcus pneumoniae) And immunized with subcutaneous route at week 0 and week 3. A vaccine alone or in alum (AlPO ₄₎ are administered in combination with the 100 ㎍ 0, 8, 40 or IL-12 of 200 ng. Conventional mouse serum (0.25%) is included as carrier protein to stabilize IL-12 at low concentrations. A control group of mice is immunized with a vaccine formulated with 100 [mu] g monophosphoryl lipid A (MPL ^R ). Mice are collected at 3 weeks (first reaction) and 5 weeks (second reaction). Serum is analyzed for IgG antibodies against Pn6B and Pb14 capsular polysaccharides by ELISA.

result

Response to PnPs6B conjugate liquid

Table 10 illustrates pulling serum IgG responses to the Pn6B component of the divalent vaccine. If the vaccine does not contain adjuvants AlPO ₄ formulation alone does not substantially react for Pn6B three weeks or the detection at all. The highest potency after a single vaccination appears to be induced by a vaccine containing 8-40 ng of MPL ^R or co-formulated IL-12 with alum. However, this level is low, ie less than 3,000. The 5 week response indicates that after the second immunization, the vaccine formulated with 40 ng of IL-12 and AlPO ₄ or MPL ^R induced the highest IgG titers to Pn6B. In the absence of alum, IL-12 in the 8 to 200 ng dose range does not enhance IgG titers to Pn6B.

The IgG subclass response to Pn6B at week 5 is shown in Table 10. It is similar in mice immunized with the formulation beksin each IgG subclass of the auxiliary station which does not contain a vaccine or AlPO ₄ (without IL-12) to go. In addition, the formulation of the vaccine with IL-12 8-200 ng of the absence of AlPO ₄ does not alter the IgG subclass response. On the other hand, this capacity of the combined IL-12 and AlPO ₄ will result in a substantially increased IgG1 and IgG2a titers for Pn6B. These potencies are similar to those obtained with the vaccine formulated with MPL ^R. IL-12 also increases the cost IgG2b and IgG3 titers induced by the vaccine formulated with AlPO _4, seems to be substantially lower than those induced by the vaccine formulated in this titer MPL ^R.

The IL-12 and the resulting increase in the formulation of AlPO ₄ In order to determine the statistical significance after, the IgG titer is determined Pn6B of each mouse in the selected group. Geometric mean titer (GMT) is shown in Table 11. The data demonstrates that the group immunized with the vaccine formulated with no adjuvant or with AlPO ₄ alone has a similar GMT for Pn6B. Formulating the vaccine with AlPO ₄ and 40 ng of IL-12 results in a 29-fold increased potency than that induced by the adjuvant-free vaccine. All data were tested by ANOVA (analysis of variance by JMP software; SAS Institute, Cary, North Carolina) and no statistically significant differences were found. Comparing the data subgroups, ANOVA shows statistically significant differences compared to the five week response induced by the vaccine containing no adjuvant and the vaccine formulated with AlPO ₄ and various doses of IL-12. Of these, the vaccine formulated with AlPO ₄ and IL-12 in the 40 ng induces a significantly higher potency than Pn6B formulated without adjuvant vaccine. As a further indication of the improved immunogenicity of the formulation, or are the seven supplements with a greater or equal Pn6B potency than 50,000 out of the 10 mice in that group only in the group vaccinated with the conjugate formulation only AlPO ₄ Compare with one mouse and two mice.

Pn6B IgG titer of each mouse group P344 P345 P346 P347 P350 P351 P352 Mouse number AlPO ₄ +200 ng IL-12 AlPO ₄ +40 ngIL-12 AlPO ₄ +8 ngIL-12 AlPO ₄ (without IL-12) 8 ng IL-12 No supplements 100 ng MPL ^R One 1,957 596,886 13,457 306,012 833,148 3,544 7,556 2 2,498 1,205 1,000,000 3,653 9,431 326 1,359,470 3 100 9,453 1,422 8,708 3,163 1,136 81 4 11,830 70,278 168,481 41,395 109,399 24,140 583,097 5 1,823 157,427 16,454 677,407 252 50,785 86,656 6 6,114 90,843 989 9,089 150,245 228 284 7 279 49,182 372,709 17,164 112 1,351 1,000,000 8 756,503 408,348 425 7,329 393 36,805 473,652 9 1,000,000 1,000,000 667,988 100 13,622 22,817 927,213 10 177 1,052,210 6,206 245 182,629 851 - GMT 4,347 103,743 22,580 9,735 10,120 3,589 55,799 A mouse with a titer of 50,000 2 7 4 2 4 One 6 Statistical comparison (ANOVA: α = 0.05) AlPO ₄ +40 ng IL-12 vs. no adjuvant:

Response to PnPs14 conjugate liquid

The IgG response to the PnPs14 component of the vaccine is shown in Table 12. Data indicate that the IL-12 of 8-40 ng dose range, and be formulated alone or AlPO _4, does not improve the response to the PnPs14 1 after primary or secondary vaccination. In addition, sub-class analysis indicates that IL-12 does not increase IgG2a titer when formulated with IL-12. In this study, MPL ^R does not have a clear adjuvant effect on the PnPs14 response observed in the previous studies, at least when pooling serum is analyzed. To obtain information on the degree of diversity of each group response, each serum is analyzed for Pn14 IgG antibody in 1/300 dilution. The results presented in Table 13 illustrate that there is a wide range of responses in each group, with a coefficient of variation (CV) ranging from 0.229 to 0.587, except for the group immunized with a vaccine containing MPL ^R with a CV of 0.051 . Thus, MPL ^{R, which} is not IL-12, may act as an adjunct to the Pn14 IgG response and seem to reduce mouse-to-mouse diversity.

Influence of IL-12 on IgG subclass responses to Pn14 in mice immunized with 2-valent PnPs6B / 14 pneumococcal conjugate liquid vaccine PnPs14 Ig ^* titer ^* At 5 weeks ^* PnPs14 IgG subclass Supplements 3 weeks 5 weeks IgG1 IgG2a IgG2b IgG3 200 ng IL-12 + AlPO ₄ 2,170 58,657 6,880 8,996 1,945 5,995 40 ng IL-12 + AlPO ₄ 1,641 53,557 8,646 3,003 3,684 2,745 8 ng IL-12 + AlPO ₄ 2,181 85,173 10,094 11,346 5,328 2,560 AlPO ₄ (without IL-12) 2,102 201,082 54,989 4,030 6,402 3,745 200 ng IL-12 849 18,293 5,769 1,582 536 799 40 ng IL-12 1,544 11,442 4,350 714 514 455 8 ng IL-12 113 12,169 5,286 354 245 330 none 509 22,601 6,080 808 618 694 100 [mu] g MPL ^R 18,616 77,106 15,745 4,275 10,205 3,916 ^* Pooling serum titer

Response of each mouse to Pn14 component of Pn6B / Pn14 2-pneumococcal conjugate liquid vaccine ^* Supplements O.D. range O.D. Average Standard Deviation Coefficient of variation AlPO ₄ + 200 ng IL-12 0.034-0.990 0.788 0.318 0.404 AlPO ₄ + 40 ng IL-12 0.457-0.948 0.771 0.176 0.229 AlPO ₄ + 8 ng IL-12 0.023-0.923 0.707 0.281 0.397 AlPO ₄ (without IL-12) 0.328-0.974 0.770 0.220 0.285 8 ng IL-12 (no alum) 0.009-0.812 0.505 0.292 0.587 No supplements 0.030-0.876 0.614 0.343 0.558 100 [mu] g MPL ^R 0.791-0.918 0.863 0.044 0.051 ^* It is tested with respect to IgG antibody to Pn14 at 1/300 dilution by ELISA on each serum

Example 5: Comparison of the effect of IL-12 in the presence or absence of alum affecting murine immune responses to univalent PnPs14-CRM197 conjugated vaccine

Research Design

BALB / c mice (8 per group) were immunized with 1 ug of PnPs14-CRM ₁₉₇ (10 ng / ml) formulated with 100 μg of AlPO ₄ or without AlPO ₄ and IL-12 or with 8, 40, 200, 1,000 or 5000 ng of IL- It is immunized with subconjunctive route at 0 week. Conventional mouse serum (0.25%) is included as carrier protein to stabilize IL-12 at low concentrations. At week 1, lymph node cell suspensions are prepared from half of each group of mice and antigen-specific cytokine production is assessed in vitro. Their spleen is also harvested and weighed. Remaining mice are harvested at 3 weeks and re-immunized with the same vaccine formulations used in the initial vaccination. Mice immunized twice every 5 weeks are drawn, the weight of these spleens is measured and their spleen cells are evaluated for cytokine production. PnPs14 and CRM ₁₉₇ IgG and IgG subclass titers are determined on pooled sera. When analysis is performed using serum from each mouse, the results are expressed in geometric mean titers (GMT).

result

Effect of IL-12 on spleen weight at 1 week after immunization

Primary a week after immunization, AlPO mouse awarded the IL-12 of 5,000 ng not a low dose of IL-12 in the absence of ₄ is significantly large spleen weight than those who receive a vaccine containing no alum or IL-12 (Table 4). Vaccine containing the AlPO ₄ is when formulated with IL-12 of from 40 to 5000 ng induces high spleen weight. Pairwise comparison shows that a vaccine formulated with 200 or 1000 ng of IL-12 and AlPO ₄ induces a greater spleen weight than those formulated with the same dose of IL-12 in the absence of AlPO ₄ . Overall, the data indicate that the formulation is an IL-12 with AlPO ₄ sikindago significantly improve the ability thereof to cause an increase in spleen weight at 1 week after the biological activity of a cytokine, namely the vaccination.

Effect of IL-12 on IgG response to PnPs14

Initially, pooled serum was analyzed for IgG antibodies to PnPs14 (Table 15). The most obvious indication of the adjuvant effect is shown after primary immunization with a vaccine containing the AlPO ₄ and 8 to 40 ng of IL-12. This formulation is compared to the mice immunized with the vaccine formulated without AlPO ₄ or IL-12 results in IgG titers increased 17 to 21 times. The combination of AlPO ₄ and IL-12 results in a greater response than when used individually: AlPO ₄ and IL-12 at a dose of 40 ng result in quadruplicate and 5-fold increased IgG titers, respectively, at 3 weeks. AlPO ₄ - Analysis of each serum from a mouse immunized with containing the vaccine (Table 16) shows the induce a 5-fold increase PnPs14 IgG titer after the first vaccination than that of the 8 ng IL-12 secondary screen alone AlPO ₄ vaccine . The difference in the titers is statistically significant. The high dose of IL-12 does not improve the response. A dose of 1,000 to 5,000 ng of IL-12 results in a significant reduction of the PnPs14 IgG titers. After the second immunization only 40 ng dose of IL-12 is AlPO ₄ - causes a significant increase in titer PnPs14 (3-fold) induced by primary vaccination.

Pooling serum data suggests that the association of AlPO ₄ with IL-12 at 8-40 ng improves IgG1 titer after primary immunization. Twice after vaccination, IL-12 are pooled serum (Table 15) and it does not increase the IgG1 titers to PnPs14 from a mouse immunized with the conjugate in the absence of AlPO ₄ as shown in the analysis of each serum (Table 17). Furthermore, the addition of from a mouse immunized with a vaccine containing the AlPO _4, 8 to 200 ng of IL-12 does not result in a higher IgG1 titers after two times vaccination (Table 17).

The most obvious effect of IL-12 is to substantially increase the PnPs14 IgG2a response at 5 weeks. This is shown in both cases the vaccine contain or be formulated without AlPO ₄ the AlPO ₄ (Table 18). In the absence of AlPO ₄ , a statistically significant increase (14-42 fold) of IgG2a GMT was obtained with IL-12 from 8 to 1,000 ng. To improve the ability of the vaccine containing - Similarly, IL-12 alone induced a capacitor 8, and 40 ng of reverse high, but the statistical, of 8-1,000 ng IL-12 is AlPO ₄ leading to IgG2a antibodies in this study. Overall, it is induced by a conjugate formulation of the highest IgG2a titers AlPO ₄ and 40 ng to IL-12. This indicates that significantly different and repeats the adjuvant activity of IL-12 enhanced by the alum and IgG2a titers induced by IL-12 of 40 ng in the absence of AlPO _4.

AgG2b and IgG3 transients are analyzed only on pooled sera (Table 15). When co-formulated with AlPO ₄ , but not in the absence thereof, an IL-12 dose ranging from 8 to 1,000 ng substantially increases IgG3 titer after primary and secondary immunization. The consistent effect of IL-12 on IgG2b titer was not specified.

Effect of IL-12 on IgG response to CRM ₁₉₇

The IgG response to CRM ₁₉₇ is also assessed by observing differences between the effects of IL-12 on protein carrier-to-polysaccharide ratio of the conjugate (Table 19). In the absence of AlPO ₄ , 40 ng of IL-12 appears to adequately increase the IgG titre to CRM ₁₉₇ after two vaccinations. However, the highest IgG potency for CRM ₁₉₇ is obtained when the vaccine is formulated with both AlPO ₄ and 8-40 ng of IL-12. The enhanced adjuvant activity of AlPO ₄ and co-formulated IL-12 resulted in an IgG titre of 40 ng of IL-12 and AlPO ₄ in 6-fold and 17-fold increments at 5 weeks, respectively, The number of times that the number increases. IL-12 causes the vaccine improves the IgG1 response to CRM _197, whether formulated with AlPO ₄ or without AlPO ₄ (table 19 and 20). IL-12 is thereby substantially increased IgG2a titers to CRM ₁₉₇ for 5 weeks after the immunization with a vaccine containing the AlPO ₄ (Table 19). Repeatly, the optimal amount of IL-12 appears to be 40 ng. Cytokines seem to increase the IgG2b titers induced by a vaccine containing the AlPO _4.

Effect of IL-12 on the cytokine profile of CRM ₁₉₇ -specific T cells

Cytokine production by splenocytes taken 2 weeks after the second vaccination (5 weeks) represents the effect of IL-12 on priming both IFN-y and IL-5 producing cells. In the case to be promoted with CRM ₁₉₇ at AlPO ₄ and IL-12 with the mouse spleen cells and IL-12 immunization in the absence is in vitro, IFN-γ is not, of the level which can detect the IL-5 IL-5 (Table 21). The formulation of the vaccine with IL-12 appears to increase the induction of IL-5-producing cells with peak activity occurring at 40 ng of cytokine. The high dose of IL-12 results in a reduced production of IL-5, and virtually no cytokines are produced by mice immunized with a co-fluid vaccine containing 1,000 to 5,000 ng of IL-12. Confirmation of IFN-y production is only detected from splenocytes of mice immunized with vaccine formulated with 5,000 ng IL-12. When the vaccine is formulated with AlPO ₄ , an addition of 8 ng of IL-12 results in priming of cells producing a sufficient amount of IFN-y, whereas only antigen-specific IL-5 production is detected in the absence of cytokines do. Priming for maximum IFN-y production appears to occur with 40-1000 ng of IL-12. The addition of 5,000 ng of IL-12 eliminates the ability of the vaccine to be primed for IL-5-producing cells.

Displayed dose of IL-12 and AlPO ₄ 100 ㎍ with or without AlPO ₄ formulation the PnPs14-CRM ₁₉₇ ball Balb a week after subcutaneous immunization with the liquid 1 ㎍ / c spleen weight of mice Supplements formulation Spleen weight (g) Group code IL-12 (ng) AlPO ₄ Average Standard Deviation P641 0 - 0.179 0.0225 P642 8 - 0.148 0.0112 P643 40 - 0.162 0.0202 P644 200 - 0.175 0.0431 P645 1,000 - 0.196 0.0068 P646 5,000 - 0.357 0.0247 P647 0 + 0.151 0.0158 P648 8 + 0.151 0.0332 P649 40 + 0.217 0.0596 P650 200 + 0.290 0.0226 P651 1,000 + 0.277 0.0919 P652 5,000 + 0.305 0.0545

Statistical comparison (ANOVA: α = 0.05)

P642, P643, P644, P645 vs. P641: Not significant

P646 vs. P641: Significant

P648 vs. P647: Not significant

P649, P650, P651, P652 vs. P647: Important

P641 vs. P647: Not significant

P644 vs. P650: Important

P642 vs. P648: Not significant

P645 vs. P651: Important

P643 vs. P649: Not significant

P646 vs. P652: Not significant

Effect of IL-12 have on the IgG Response to PnPs14 AlPO ₄ in the PnPs14-CRM ₁₉₇ ball mouse immunized with a liquid formulation PnPs14 IgG GMT (fold increase) group IL-12 (ng) AlPO ₄ 3 weeks 5 weeks P647 0 + 3,037 27,027 P648 8 + 16,68 (5, 5) 55,855 (2,1) P649 40 + 6,667 (2,2) 88,271 (3,4) P650 200 + 2,333 (0,8) 57,076 (2,1) P651 1,000 + 611 (0,2) 30,886 (1,1) P652 5,000 + 617 (0,2) 10,989 (0,4)

Statistical comparison (ANOVA: α = 0.05)

3 weeks

P648 vs. P647: NOTES

P651 vs. P647: NOTES

P649, P650, P652 vs. P647: Not significant

5 weeks

P649 vs. P647: NOTES

P648, P650, P651 vs. P647: Not significant

IL-12 and AlPO with different capacities to ₄ or AlPO ₄ formulation without the PnPs14-CRM ₁₉₇ conjugate vaccine twice from immunized mice IgG1 titer PnPs14 Protective formulations Group code IL-12 (ng) AlPO ₄ IgG1 GMT (geometric mean titer) P641 0 - 9,492 P642 8 - 5,964 P643 40 - 14,028 P644 200 - 4,628 P645 1,000 - 5,815 P646 5,000 - 1,757 P647 0 + 15,283 P648 8 + 35,730 P649 40 + 31,855 P650 200 + 34,166 P651 1,000 + 15,347 P652 5,000 + 4.022

Statistical comparison (ANOVA: α = 0.05)

P642, P643, P644, P645, P646, P647, P651 vs. P641: Not significant

P648, P649, P650 vs. P641: Important

P648, P649, P650, P651 vs. P647: Not significant

P652 vs. P647: NOTES

IL-12 and AlPO ₄ in a different content of AlPO ₄ or a formulation without the PnPs14-CRM ₁₉₇ conjugate vaccine two mice immunized once PNPS14 IgG2a titer Group code IL-12 (ng) AlPO ₄ IgG2a GMT at 5 weeks (fold increase ^* ) P641 0 - 97 P642 8 - 1,418 (14,6) P643 40 - 1,509 (15,6) P644 200 - 2,228 (23,0) P645 1,000 - 4,126 (42,5) P646 5,000 - 289 (3, 0) P647 0 + 806 P648 8 + 6,841 (8, 5) P649 40 + 13,252 (16,4) P650 200 + 4, .740 (5,9) P651 1,000 + 3,291 (4,1) P652 5,000 + 368 (0,5) ^* Compared with a vaccine that does not contain IL-12

Statistical comparison (ANOVA: α = 0.05)

P642, P643, P644, P645 vs. P641: Important

P646 vs. P641: Not significant

P648, P649 vs. P647: Important

P650, P651, P652 vs. P647: Not significant

P643 vs. P649: NOTES

IgG1 titers to IL-12 and AlPO the CRM ₁₉₇ in BalB / c mice immunized with PnPs14-CRM ₁₉₇ conjugate formulated in ₄ Group code IL-12 (ng) AlPO ₄ IgG1 GMT Multiplication P641 0 - 317 - P642 8 - 1,136 3.6 P643 40 - 9,141 28.8 P644 200 - 1,627 5.1 P645 1,000 - 100 0.3 P646 5,000 - 174 0.6 P647 0 + 22,061 _ P648 8 + 119,130 5.4 P649 40 + 73,226 3.3 P950 200 + 14,391 0.7 P651 1,000 + 33,468 1.5 P652 5,000 + 317 0.01

Statistical comparison (ANOVA: α = 0.05)

P643, P644 vs. P641: Important

P642, P645, P646 vs. P641: Not significant

P648, P649, P650, P651 vs. P647: Not significant

P642 to P648, P643 to P649, P644 to P650, P645 to P651: Important

Example 6: Influence of IL-12 / AlPO ₄ on humoral response to Bimu atom pneumococcal conjugate liquid vaccine

Research Design

Assessment of the effect of IL-12 on IgG responses to pneumococcal conjugate liquid vaccines is extended to a non-mAb vaccine consisting of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. Swiss Webster mice are immunized with 0.1, 1 or 5 [mu] g vaccine (hydrocarbon weight) at 0 and 3 weeks. Vaccines alone, AlPO ₄ is administered with the blend with (100 ㎍) with or 50, 200 or 1,000 ng of IL-12 AlPO _4. Conventional mouse sera are not included in the vaccine. IgG responses to serotype 4, 6B, 9V, 14, 18C and carrier protein CRM ₁₉₇ are assessed by ELISA at 5 weeks (ie, 2 weeks after secondary immunization).

result

Response to CRM ₁₉₇ at 5 weeks

The addition of IL-12 to a vaccine containing AlPO ₄ results in a dose-dependent increase of IgG2a and IgG2b antibodies to CRM ₁₉₇ . This occurs at all doses of the tested conjugate (Table 22). The increased IgG2a potency is evident in mice given 50 ng and up to 1,000 ng cytokines. This is compared with other studies in which the highest IgG2a titer is obtained with 40-100 ng of cytokine added to the alum-basic vaccine and the high content of IL-12 results in a reduced immune response. The reasons for the differences in dose dependencies between studies were not revealed. This may be due to the difference in vaccine, that is, the normal mouse serum included in the vaccine in the previous test is omitted in order to stabilize the cytokine at the polyvalent single or low concentration.

Responses to pneumococcal polysaccharides

The formulation of the Bimu valent vaccine with AlPO ₄ improves the IgG response against a number of serotypes, including PnPS4, PnPs6B, PnPs9V and PnPs14, particularly when low doses of the conjugate (0.1 μg) are used (Table 24-27) . The addition of IL-12 does not appear to further improve the IgG response to these serotypes. However, the addition of IL-12 of 50 or 1,000 ng of the 5 ㎍ vaccine containing the AlPO ₄ in the case of PnPs18C reaction is higher proportion of mice having a high geometric mean IgG titers and 10,000 or more PnPs18C IgG titers to the serotypes (Table 23). The responses to PnPs1, 5, 19F and 23F were not evaluated.

AlPO the bimu atoms vaccine containing ₄ The addition of IL-12 the capacity of the IgG2a titers for PnPs4, PnPs6B, PnPs9V PnPs14 and - resulting in a dependent increase (Table 24-27). In general, the increase in IgG2a is compared to the CRM ₁₉₇ response with the highest titre obtained with 1,000 ng of IL-12. Compared with experiments using monovalent PnPs14 conjugated liquid or divalent PnPs6B / PnPs14 vaccine, a 50 ng dose of IL-12 has little or no effect on the IgG2a response to these serotypes. The exception is the IgG2a response to PnPs14, as this dose of cytokines appears to enhance the response to this serotype (Table 27).

Collectively, this study explains that IL-12 will enhance the complement-bound IgG2a antibody subclass response to the multiple pneumococcal serotype present in the multivalent vaccine.

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Serum collected at 5 weeks is analyzed for IgG antibodies against CRM ₁₉₇ .

Influence of IL-12 on the response to PnPs18C in mice immunized with 5 ㎍ of Bimeta pneumococcal conjugate liquid vaccine formulated with AlPO ₄ Vaccine formulation PnPs18C reaction Bulky liquid capacity (㎍) IL-12 (ng) AlPO ₄ (㎍) IgG titers (GMT) Gt; 10,000 < / RTI > (n = 5) 5 0 0 2,933 One 5 50 100 23,725 4 5 1,000 100 48,375 5

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Each mouse serum collected at 5 weeks is analyzed for IgG antibody against PnPs18C.

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Serum collected at 5 weeks is analyzed for IgG antibodies against PnPs4.

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Serum collected at 5 weeks is analyzed for IgG antibodies against PnPs6B.

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Serum collected at 5 weeks is analyzed for IgG antibodies against PnPs9V.

Mice are immunized at 0 and 3 weeks with the indicated doses of Bimetallic Streptococcus pneumoniae liquid vaccine. The conjugate is formulated with AlPO ₄ (100 μg) alone or with AlPO ₄ and IL-12. Serum collected at 5 weeks is analyzed for IgG antibodies against PnPs14.

Example 7: Influence of IL-12 and AlPO ₄ on immune responses to meningococcal form vaccine (menC)

Research Design

This study evaluates IL-12 as a vaccine against meningococcal form C (menC). Swiss Webster mice are immunized at 0 and 3 weeks with 0.1 μg or 1 μg of MenC conjugate formulated with AlPO ₄ (100 μg) alone or as a conjugate of AlPO ₄ and IL-12 (50 ng). Conventional mouse serum is not added to the vaccine. Mice were collected at 3 and 5 weeks and serum was analyzed by ELISA for IgG antibodies against MenC polysaccharides.

result

When immunized with a high dose of conjugate, an equivalent menC IgG titre is produced irrespective of the adjuvant formulations. However, the addition of IL-12 / AlPO ₄ in the vaccine results in a higher IgG2a titers to the polysaccharide than when the formulation without adjuvant, or with AlPO ₄ (no IL-12).

In mice immunized with a low dose of conjugate, a high menC potency was obtained when the vaccine was formulated with AlPO ₄ (Table 28). Addition of IL-12 to adjuvant does not increase the total IgG titer but results in a> 10-fold increase in IgG2a antibody. These data are combined IL-12 and AlPO ₄ complement for menC danggong liquid vaccine - shows that to improve the inductive coupling of the IgG subclasses.

Example 8 Influence of IL-12 and AlPO ₄ on Immune Response to Hemophilus Influenza Type b Urinary Fluid Vaccine (HbOC)

Research Design

This study evaluates IL-12 as a vaccine against Haemophilus influenzae b. Swiss Webster mice (10 per group) are immunized with 0.1 μg or 1.0 μg of an adult liquid vaccine consisting of capsular polysaccharides of Haemophilus influenzae form (HibPs) formulated in CRM ₁₉₇ at 0 and 3 weeks. The vaccine (HbOC) is administered alone or in combination with AlPO ₄ (100 μg) or in combination with a mixture of IL-12 (50 ng) and AlPO ₄ . Conventional mouse serum is not added to the vaccine. Mice are collected at 3 and 5 weeks. The antibody response to HibPs is measured using the Farr assay, which measures all antibodies that bind to the saccharide regardless of isotype, IgM, IgG and IgA. The IgG subclass response is measured by ELISA. Additionally, IgG and IgG subclass responses to CRM ₁₉₇ are also measured by ELISA.

result

Reverse transcription of anti-HibPs antibodies in pooled sera (primary response) collected at 3 weeks (primary response) Immunization with vaccine alone or in formulated with AlPO ₄ or IL-12 and AlPO ₄ , irrespective of the volume of conjugate used for immunization (Table 29). Analysis of pooled sera collected at 5 weeks shows that the mice immunized with 1 [mu] g of HbOC with IL-12 and alum resulted in at least a 10-fold increase in anti-HibPs as compared to that given without alum or without adjuvant (Table 30). However, analysis of each mouse serum indicates that this is due to a single mouse with a titer of approximately 10,000 [mu] g / ml. There is no evidence of an enhanced HibPs response due to IL-12 when results are expressed as geometric mean titres. The IgG subclass response to HibPs is assessed in pooled sera by ELISA. The formulation of IL-12 and AlPO ₄ are likely to increase to three times the IgG2a titers in mice immunized with the conjugate of 1 ㎍. However, this is not different from the obtained activity, and as a secondary vaccine with AlPO ₄ alone. In mice immunized with 0.1 μg of HbOC, IL-12 and AlPO ₄ did not enhance IgG2a titers to HibPs. The fact that the IL-12 / AlPO ₄ adjuvant combination is active is indicated by the analysis of the anti-CRM ₁₉₇ response (Table 31) in which the increased IgG2a titer for the carrier protein is shown in mice immunized with conjugated volume.

Anti-HibPs antibody response of mice immunized with HbOC formulated with IL-12 and AlPO ₄ Anti-HibPs antibody reaction ([mu] g / ml) Vaccine formulation 3 weeks 5 weeks HbOC (㎍) IL-12 ( ng) AlPO 4 (㎍) Pooling serum Pooling serum GMT ^* 1.0 50 100 9.73 469.16 26.92 0 100 10.04 42.55 21.30 0 0 5.12 33.19 2.25 0.1 50 100 3.18 30.95 ND 0 100 4.06 15.11 ND 0 0 3.03 14.05 ND

Influence of IL-12 on IgG subclass response to HbOC Vaccine formulation 5-week anti-HibPs IgG subclass response (ELISA end-point titers) HbOC ([mu] g) IL-12 (ng) AlPO4 ([mu] g) IgG1 IgG2a 1.0 50 100 754,745 26,899 0 100 122,637 12,880 0 0 73,114 8,570 0.1 50 100 46,673 17,290 0 100 68,176 14,971 0 0 35,237 11,418

Anti-CRM ₁₉₇ IgG response of mice immunized with HbOC formulated with IL-12 and AlPO ₄ Vaccine formulation At 5 weeks anti-CRM ₁₉₇ response _{HbOC IL-12 AlPO 4 (㎍} ) (ng) (㎍) IgG IgG1 IgG2a IgG2b 1.0 50 100 1,775,700 681,944 39,672 40,527 0 100 2,221,780 818,557 19,010 32,672 0 0 3,979,530 1,466,010 8,059 15,961 0.1 50 100 761,027 292,448 38,258 21,008 0 100 891,251 346,728 6,546 14,832 0 0 874,805 151,397 1,899 3,517

Equivalent method

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many methods that are equivalent to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (56)

A vaccine composition comprising a pneumococcal antigen, an adjuvant of interleukin-12 and a mixture of minerals in suspension, and optionally a physiologically acceptable vehicle. 12. The vaccine composition of claim 1, wherein the interleukin-12 is adsorbed onto a mineral suspension. The vaccine composition of claim 1, wherein the interleukin-12 is human interleukin-12. The vaccine composition of claim 1, wherein the mineral in the suspension is an aqueous suspension of alum. 5. The vaccine composition of claim 4, wherein the alum is aluminum hydroxide or aluminum phosphate. The vaccine composition of claim 1, wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and combinations thereof. The vaccine composition according to claim 1, wherein the pneumococcal antigen is conjugated to a carrier molecule. 8. The vaccine composition of claim 7, wherein the carrier protein is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin, and non-toxic modifications thereof. 9. The vaccine composition of claim 8, wherein the carrier molecule is CRM ₁₉₇ . Comprising administering to the mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant of interleukin-12 and a mineral in a suspension, optionally comprising a physiologically acceptable vehicle, A method of inducing a reaction. 11. The method of claim 10 wherein the interleukin-12 is adsorbed onto a mineral suspension. 11. The method of claim 10, wherein the interleukin-12 is human interleukin-12. 11. The method of claim 10, wherein the mineral in the suspension is an aqueous suspension of alum. 14. The method of claim 13, wherein the alum is aluminum hydroxide or aluminum phosphate. 11. The method of claim 10, wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and combinations thereof. 11. The method of claim 10 wherein the pneumococcal antigen is conjugated to a carrier molecule. 17. The method of claim 16, wherein the carrier molecule is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin and non-toxic modifications thereof. 18. The method of claim 17, wherein the carrier molecule is CRM ₁₉₇ . Comprising administering to the mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant of interleukin-12 and a mineral in a suspension, optionally comprising a physiologically acceptable vehicle, -γ reaction. An effective amount of an immunogenic composition comprising a mixture of minerals in a parenteral antibiotic antigen, interleukin-12 supplement and suspension, optionally comprising a physiologically acceptable vehicle, in a mammalian host, Lt; RTI ID = 0.0 > complement-binding < / RTI > A pneumococcal antigen, an adjuvant of interleukin-12, and a mixture of minerals in suspension, optionally comprising a physiologically acceptable vehicle. 22. The immunogenic composition of claim 21, wherein the interleukin-12 is adsorbed onto a mineral suspension. 22. The immunogenic composition of claim 21, wherein the interleukin-12 is human interleukin-12. 22. The immunogenic composition of claim 21, wherein the mineral in the suspension is an aqueous suspension of alum. 25. The immunogenic composition of claim 24, wherein the alum is aluminum hydroxide or aluminum phosphate. 22. The immunogenic composition of claim 21 wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and combinations thereof. 22. The immunogenic composition of claim 21, wherein the pneumococcal antigen is conjugated to a carrier molecule. 28. The composition of claim 27, wherein the carrier molecule is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin, and non-toxic modifications thereof. 29. The immunogen composition according to claim 28, wherein the carrier molecule is CRM < RTI ID = 0.0 > ₁₉₇ . A vaccine composition comprising a mixture of a meningococcal antigen, an adjuvant of interleukin-12 and a mineral in a suspension, and optionally a physiologically acceptable vehicle. 31. The vaccine composition of claim 30, wherein the interleukin-12 is adsorbed onto a mineral suspension. 31. The vaccine composition of claim 30, wherein the interleukin-12 is human interleukin-12. 31. The vaccine composition of claim 30, wherein the mineral in the suspension is an aqueous suspension of alum. 34. The vaccine composition of claim 33, wherein the alum is aluminum hydroxide or aluminum phosphate. 31. The vaccine composition according to claim 30, wherein the meningococcal antigen is an adult meningitis C type capsule polysaccharide. 31. The vaccine composition of claim 30, wherein the meningococcal antigen is conjugated to a carrier molecule. 37. The vaccine composition of claim 36, wherein the carrier molecule is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin, and non-toxic modifications thereof. 38. The vaccine composition of claim 37, wherein the carrier molecule is CRM < RTI ID = 0.0 > ₁₉₇ . Immunization against meningococcal antigens, comprising administering to the mammalian host an effective amount of a vaccine composition comprising a mixture of minerals in a suspension amount of meningococcal antigen, interleukin-12 and optionally a physiologically acceptable vehicle A method of inducing a reaction. 40. The method of claim 39, wherein the interleukin-12 is adsorbed onto the mineral suspension. 40. The method of claim 39, wherein the interleukin-12 is human interleukin-12. 40. The method of claim 39, wherein the mineral in the suspension is an aqueous suspension of alum. 43. The method of claim 42 wherein the alum is aluminum hydroxide or aluminum phosphate. 40. The method of claim 39, wherein the meningococcal antigen is a meningococcal bacterial C-type capsule polysaccharide. 41. The method of claim 39, wherein the meningococcal antigen is conjugated to a carrier molecule. 46. The method of claim 45, wherein the carrier molecule is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin and non-toxic modifications thereof. 47. The method of claim 46, wherein the carrier molecule is CRM ₁₉₇ . An immunosuppressive agent, a meningococcal antigen, an adjuvant of interleukin-12, and a mixture of minerals in suspension, optionally comprising a physiologically acceptable vehicle. 49. The immunogenic composition of claim 48, wherein the interleukin-12 is adsorbed onto a mineral suspension. 49. The immunogenic composition of claim 48, wherein the interleukin-12 is human interleukin-12. 49. The immunogenic composition of claim 48, wherein the mineral in the suspension is an aqueous suspension of alum. 52. The immunogenic composition of claim 51, wherein the alum is aluminum hydroxide or aluminum phosphate. 49. The immunogenic composition of claim 48, wherein the meningococcal antigen is a meningococcal bacterial form C capsular polysaccharide. 49. The immunogenic composition of claim 48, wherein the meningococcal antigen is conjugated to a carrier molecule. 55. The immunogenic composition of claim 54, wherein the carrier molecule is selected from the group consisting of a tetanus toxin, a diphtheria toxin, a pertussis toxin and non-toxic modifications thereof. 56. The immunogenic composition of claim 55, wherein the carrier molecule is CRM < RTI ID = 0.0 > ₁₉₇ .