JP2002502882A - Pneumococcal and meningococcal vaccines formulated with interleukin-12 - Google Patents

Abstract

  (57) [Summary] The present invention relates to a vaccine composition comprising a mixture of an antigen, such as a pneumococcal or meningococcal antigen, and interleukin IL-12, which may be adsorbed on the mineral in suspension. The pneumococcal or meningococcal antigen may be conjugated to a carrier molecule. These vaccine compositions modulate a protective immune response to the antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The immune system uses many mechanisms to attack pathogens; however, not all of these mechanisms are necessarily activated after immunization. The protective immunity induced by vaccination depends on the ability of the vaccine to elicit an appropriate immune response to resist or eliminate the pathogen. Depending on the pathogen,
This may require a cell-mediated and / or humoral immune response.

The current paradigm for the role of helper T cells in the immune response is that they can be separated into subsets based on the cytokines they produce, and
A characteristic of the unique cytokines observed in these cells is that they determine their function. This T cell model has two major subsets: IL-2 and interferon gamma (IFN) that increase both cellular and humoral immune responses.
-Γ) producing TH-1 cells, and IL-4 increasing humoral immune response
, IL-5 and IL-10 producing TH-2 cells (Mossman (M
osmann) et al. Immunol. 126: 2348 (1986)). To obtain a stronger immune response in the immunized organism, and to pathogens with antigens
It is often desirable to increase the immunogenic potential of an antigen to increase host resistance to the agent. Substances that increase the immunogenicity of the antigen with which they are administered are known as adjuvants. For example, certain lymphokines have been shown to have adjuvant activity, thereby enhancing the immune response to antigens (Nencioni et al., J. Immunol. 139: 800-80).
4 (1987); EP 285441 to Howard et al.).

SUMMARY OF THE INVENTION The present invention relates to a vaccine composition comprising a mixture of one or more pneumococcal or meningococcal antigens, interleukin IL-12, and a mineral in suspension. . IL-12 can either be adsorbed on the mineral suspension or simply mixed with them. In one particular aspect of the invention, the IL
-12 is adsorbed on a mineral suspension such as alum (eg, aluminum hydroxide or aluminum phosphate). These vaccine compositions modulate a protective immune response to the antigen; that is, they improve quantitatively and qualitatively the antibody response of the vaccinated host, and It is possible to quantitatively increase cell-mediated immunity for the response. In one particular embodiment of the invention, the antigen is a pneumococcal or meningococcal antigen; the antigen is optionally linked to a carrier molecule as in a pneumococcal or meningococcal glycoconjugate .

[0004] The studies described herein show that IL-12 can be treated with aluminum phosphate (AlPO). _Four 4) can modify the humoral response of mice immunized with a pneumococcal and meningococcal glycoconjugate vaccine formulated with). Specific lungs exemplified herein
The serotypes of streptococcal polysaccharides are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F.
And 23F (Pn1, Pn4, Pn5, Pn6B, Pn9V, Pn14, Pn
18C, Pn19F and Pn23F), and the meningococcal polysaccharide is type C (
Men C). These serotypes, however, limit the scope of the invention
Should not be interpreted as Because the serotypes of other pneumococci and meningococci are
It is also suitable for use herein. In addition, the CRMs exemplified herein ₁₉₇ It will be apparent to those skilled in the art that conjugation to a carrier molecule, such as a protein, is optional, depending on the immunogenicity of the selected pneumococcal or meningococcal antigen.

[0005] Dose of IL-12, ranging from about 8 ng to about 1,000 ng, can be adjusted to IgG1, IgG2a, IgG for Pn14 or Pn6B adsorbed on alum.
2b and IgG3 responses were increased. In addition, they include Pn4 and Pn9
Increased IgG2a response to V. A dose of about 5,000 ng of IL-12 will give a total IgG titer against Pn14, and especially IgG1 and IgG2b
Significantly reduced the titer.

[0006] The present invention also relates to a method of preparing an immunogenic or vaccine composition comprising a mixture of antigen and IL-12 with minerals in suspension. Above all, I
L-12 is adsorbed on the mineral suspension. The present invention also relates to the administration of an effective amount of a vaccine composition comprising a mixture of antigen, IL-12 and a mineral in suspension in a physiologically acceptable solution to a mammalian, e.g., human or primate, host. A method for deriving or expanding IFN-γ producing T cells and complement binding IgG antibodies of a vaccine for a protective immune response, comprising: Above all, I
L-12 is adsorbed on the mineral suspension.

DETAILED DESCRIPTION OF THE INVENTION [0007] The work described herein has been directed to alum-based pneumococcal vaccines, especially serotype 1 for increasing the proportion of complement-fixed IgG2a and IgG2b antibodies.
4 and serotype 6B pneumococcal glycoconjugate vaccines, and meningococcal vaccines,
In particular, it shows the ability of IL-12 to increase the immune response to type C. As used herein, the PnPs-14-CRM ₁₉₇ vaccine is a serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant (cross-reacting substance) of diphtheria toxoid designated CRM _197. And PnPs6B-CR
M ₁₉₇ vaccine comprises pneumococcal polysaccharide serotype 6B which is coupled to the CRM _197. IL-12 is a potent adjuvant for pneumococcal vaccine in mice, MPL ™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc.).
rch, Inc. ), Hammitton, Montana). In a separate experiment performed in Balb / c mice, the effect of IL-12 on the cytokine characteristics of CRM-specific T cells induced by an exemplary conjugate vaccine on alum was examined.

[0008] IL-12 is produced by a variety of antigen presenting cells, mainly macrophages and monocytes. It is a critical factor in the induction of TH-1 cells from naive T cells. The production of IL-12, or the ability to respond thereto, has been shown to be critical in the development of a protective TH-1-like response during, for example, parasitic infections, most notably leishmaniasis. (Scott et al., US Pat. No. 5,571,515). IL-
Twelve effects are mediated by IFN-γ produced by NK cells and T helper cells. Interleukin-12 (IL-12), originally called natural killer cell stimulating factor, is a heterodimeric cytokine (Kobayashi (Kobayashi)
ashi) et al. Exp. Med. 170: 827 (1989)). Expression and isolation of IL-12 protein in recombinant host cells was described on May 17, 1990.
It is described in International Patent Application No. WO 90/05147, published today.

[0009] The studies described herein demonstrate the utility of IL-12 as an adjuvant in pneumococcal or meningococcal vaccines, and especially pneumococcal or meningococcal glycoconjugate vaccines. Accordingly, the present invention relates to a vaccine composition comprising a mixture of such an antigen, IL-12 and a mineral in suspension. In one particular aspect of the invention, IL-12 is adsorbed on a mineral suspension such as alum (eg, aluminum hydroxide or aluminum phosphate). These vaccine compositions modulate a protective immune response to the antigen; that is, the vaccine composition is able to elicit complement-binding antibodies of the vaccinated host for a protective response to the pathogen. . In one particular embodiment of the invention, the antigen is a pneumococcal antigen, especially a pneumococcal polysaccharide; the pneumococcal antigen is optionally conjugated to a carrier molecule such as a pneumococcal glycoconjugate. . The serotypes of certain pneumococcal polysaccharides exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C,
19F and 23F; however, these serotypes are not to be construed as limiting the scope of the invention. Because other serotypes are also suitable for use herein.

In another embodiment of the invention, the antigen is a meningococcal antigen, especially a meningococcal polysaccharide; the meningococcal antigen is optionally a meningococcal glycoconjugate Is conjugated to the carrier molecule. Type C meningococcus (Neisseria mening)
itis) are exemplified herein; however, this form is not to be construed as limiting the scope of the invention. Because other types are also suitable for use herein.

[0011] IL-12 can be obtained from several suitable sources. It is a recombinant D
For example, the gene encoding human IL-12 may be cloned and expressed in a host system to allow for the production of large amounts of pure human IL-12. Biologically active subunits or fragments of IL-12 are also useful in the present invention. Commercial sources of recombinant human and mouse IL-12 are from Genetics Institute Inc.
nstate, Inc. ) (Cambridge, MA).

An antigen of the invention, such as a pneumococcal or meningococcal antigen, or a pneumococcal or meningococcal glycoconjugate, can be used to elicit an immune response to the antigen in a mammalian host. For example, the antigen is a serotype 14 or 6B pneumococcal polysaccharide,
Or it may be a part thereof that retains the ability to stimulate an immune response. Additional suitable antigens include other encapsulated bacteria and their complexes, secreted toxins and polysaccharides from outer membrane proteins.

The method comprises immunizing a vaccine composition comprising a mixture of an antigen, such as a pneumococcal antigen or a pneumococcal complex, and an adjuvant amount of IL-12 adsorbed on the mineral in suspension. Administering to a mammal, eg, a human or primate, a chemically effective amount.

[0014] As used herein, an "immunologically effective" dose of a vaccine composition is a dose suitable for eliciting an immune response. The particular dosage of IL-12 and antigen can depend on the age, weight and medical condition of the mammal to be treated, and the mode of administration. Suitable doses will be readily determined by one skilled in the art. The vaccine composition can optionally be administered in a pharmaceutically or physiologically acceptable vehicle, such as physiological saline or an ethanolic polyhydric alcohol such as glycerol or propylene glycol.

[0015] The vaccine composition optionally comprises a vegetable oil or an emulsion thereof, a surfactant such as hexadecylamine, octadecylamino acid ester, octadecylamine, lysolecithin, dimethyldioctadecyl ammonium bromide, N
, N-dicoctadecyl-N ', N'-bis (2-hydroxyethylpropanediamine), methoxyhexadecylglycerol, and pluronic polyols (p
luronic polyols); polyamines such as pyran, dextran sulfate, poly IC,
Carbopol, peptides; eg muramyl dipeptide, dimethylglycine, tuftsin; immunostimulatory complexes; oil emulsions; liposaccharides such as MPL ™ and additional adjuvants such as mineral gels. The antigens of the present invention can also be incorporated into liposomes, cocheletes, biodegradable polymers such as polylactides, polyglycolides and polylactide coglycolides, or ISCOMS (immunostimulating complexes) and supplement active ingredients. May also be used. The antigens of the present invention may also be co-administered with bacterial toxins and their attenuated derivatives. The antigen of the present invention can also be used for IL-2, IL-3, IL-15, IFN-γ.
And may be co-administered with other lymphokines, including but not limited to GM-CSF.

The vaccine includes parenteral, intradermal, transdermal (such as through the use of a delayed release polymer), intramuscular, intraperitoneal, intravenous, subcutaneous, oral and nasal route of administration but Can be administered to humans or animals by a variety of routes, without limitation. The amount of antigen used in such vaccines can vary depending on the identity of the antigen. The adjustment and manipulation of established dosage ranges to be used with traditional carrier antigens for adaptation to the present vaccine is well within the ability of those skilled in the art. The vaccines of the present invention are intended for use in the treatment of both immature and adult warm-blooded animals, and especially humans. Typically, IL-12 and antigen can be co-administered; however, in some instances, those skilled in the art will recognize that IL-12 can be administered in close but not before vaccination with the antigen. Or it will be appreciated that it can be administered subsequently.

The pneumococcal and meningococcal antigens of the invention can be conjugated to a carrier molecule to modulate or enhance an immune response. Suitable carrier proteins include bacterial toxins that are made safe by chemical or genetic means and immunologically effective as carriers for administration to mammals. Examples include pertussis, diphtheria and tetanus toxoids, and non-toxic mutant proteins such as the non-toxic mutant CRM ₁₉₇ of diphtheria toxoid (cross-reactant (CRM)). Fragments of naturally occurring toxins or toxoids containing at least one T cell epitope are also useful as carriers for antigen, as are outer membrane protein complexes. Methods for making conjugates of pneumococcal antigens and carrier molecules are known in the art and include, for example, Dick and Burret,
Contrib Microbiol Immunol. 10: 48-114 (
Cruise JM, Lewis RE Jr, eds .; Basel, Krager (1989)) and US Patent No. 5,360,897 (Anderson et al.).

The adjuvant effect of IL-12 has a number of important implications. The adjuvant properties of IL-12 can increase the concentration of protective functional antibodies produced against the antigen in the vaccinated organism. The use of IL-12 as an adjuvant
It may enhance the ability of weakly antigenic or poorly immunogenic antigens to elicit an immune response. It can also provide for safer vaccination if the antigen is toxic at concentrations normally required for effective immunization. By reducing the amount of antigen, the risk of a toxic reaction is reduced.

Typically, vaccination regimes require administration of the antigen over a period of weeks or months to stimulate a “protective” immune response. A protective immune response is an immune response sufficient to protect the immunized organism from productive infection by the particular pathogen or pathogens to which the vaccine is directed.

[0020] performed as shown in the example, a vaccine formulated with alum comprising pneumococcal polysaccharide serotype 14 or serotype 6B conjugated to IL-12 and CRM _{1 97} adsorbed onto AlPO ₄ ( 0.2 μg of IL-12 was expressed in both Balb / c and Swiss Webster mice in IgG2a and IgG3.
Were essentially increased, but had little or no effect on IgG1. Enhancement of IgG2b on Pn14 is seen in Swiss Webster mice; 0.2 μg of IL
-12 responds to the IgG subclass response to Pn14 by 25 μg of MPL (
Has the same effect as MPL (TM), suggesting that IL-12 is at least 100 times more biologically active than MPL (TM) in this regard. As expected from the distribution of IgG subclasses, especially the enhanced IgG2a response, 0.2 μg of I
The opsonophagocytic activity of the antiserum against Pn14 pneumococcus from mice receiving L-12 is higher than that of the control, and that of mice immunized with a vaccine formulated with much higher amounts of MPL ™. Was equivalent to the one.

[0021] Briefly, IgG2a and IgG2b antibodies are very efficient at activating the complement system, while IgG1 antibodies are not. The complement system consists of a series of plasma proteins that together form a large molecular complex around IgG2a or IgG2b bound to an antigen (eg, a bacterium). The attachment of this complex on the surface of bacteria can either puncture cell membranes (bactericidal activity) or take up and kill bacteria (opsonophagocytosis) (the multinuclear leukocytes (PMN) used in this study). like)
Facilitating bacterial recognition by phagocytic cells results in bacterial killing.

Increasing the dose of IL-12 greatly reduced IgG1 and IgG2b responses. Reduction of these immunoglobulin subclasses was observed in the egg lysozyme (HEL) system (Buchanan, Van Cleave and Metzger, abstract # 1945;
9th International Conference on Immunology (9th International Congres)
s of Immunology (1995)), but not simply due to a change in the dynamics of the antibody response. Because these subclasses decreased at all time points tested. The effect on IgG1 is that B cells are switched to this subclass by IL-4 (TH whose production is inhibited by IL-12).
-2 cytokines). However I
No reduction in gG2b was expected. Because, in previous studies, increased levels of IgG2b correlated with the presence of TH-1 like T cells. It is likely that cytokines other than or in addition to IFN-γ are involved in the regulation of IgG2b. For example, German (Eur. J. I.)
mmunol. 25: 823-829 (1995)) found that treatment of mice with anti-IFN-γ inhibited the ability of IL-12 to enhance the IgG2a response but not IgG2b. Other studies have implicated TGF-β as a key factor in the induction of IgG2b (J. Stavneze).
r), J. et al. Immunol. 155: 1647-1651 (1995)). Without wishing to be bound by theory, it is believed that high doses of IL-12
-It may be possible to affect β production or responsiveness to it.

IFN-γ induces IgG2a antibodies against T-dependent protein antigens (Finkelman and Holmes, Annu. R
ev. Immunol. 8: 303-33 (1990)), and the response of IgG3 to T-independent antigens (Snapper et al., J. Exp. M.
ed. 175: 1367-1371 (1992)). An enhanced IFN-γ response was consistently found after a single vaccination with a vaccine containing IL-12 and AlPO ₄ (PnPs-14-CRM ₁₉₇ ) and after a boost. The effect of IL-12 on the TH-2 cytokines IL-5 and IL-10 appears to be dependent upon the lymphoid cells being harvested after vaccination, and likely to be specific cytokines. Exogenous IL-12
Completely abolished antigen-specific IL-5 and IL-10 production by lymph node cells (LNC) harvested one week after primary vaccination. After the second vaccination, differences were seen between these two cytokines; IL-5 production by either LNC or splenocytes was completely abolished by 1 μg of IL-12 in the vaccine, However, IL-10 production was not significantly affected after booster stimulation. It is unclear whether these differences are due to the composition of the cultures at different times or reflect the expansion of the TH-2-like population upon subsequent re-vaccination. The latter possibility is described by Wolf and coworkers (Bliss et al., J. Immunol. 156: 887-).
894 (1996)).
B already immunized with a vaccine containing No. 12 and boosted with a soluble antigen
This shows that it can be recovered from alb / c mice. In their study, IL-4 was
-12 was detected even when included in the second vaccine. The presence of TH-2 cytokines after boosting could not reduce secondary IgG1 responses below control levels (complex vaccine on alum), even at high levels of IL-12 in Balb / c mice It may explain the reason. Unlike Balb / c mice, high doses of IL-12 severely inhibited the IgG1 response in Swiss Webster mice. It is unclear whether this is associated with reduced production of TH-2 cytokines after the second vaccination.

In the present study, IL-12 either showed only immunomodulatory activity, or behaved as both a “classical” adjuvant and an immunomodulator depending on the vaccine. In studies with PnPs14-CRM ₁₉₇ , the IgG response to the vaccine (especially the primary response) was not essentially increased by the presence of cytokines, but was not effective for certain subclasses, namely IgG2a.
And IgG3 increased while others remained unchanged or decreased. Thus, IL-12 is useful for altering the humoral response to an already immunogenic vaccine. In these studies, the adjuvant activity of IL-12 can be masked by the presence of alum, a sufficient adjuvant of the PnPs-14 complex by itself and highly immunogenic. The adjuvant properties of IL-12 may be better demonstrated in the absence of alum by reducing the dose of the conjugate or using a poorly immunogenic conjugate. Therefore, further evaluation was made using IL-12 in the presence and absence of alum using the PnPs6B conjugate vaccine, which is smaller and more immunogenic than the PnPs-14 conjugate vaccine in Swiss Webster mice. It was carried out.

An additional study was designed to address the issue of IL-12 adjuvant activity for the poorly immunogenic pneumococcal complex. The Pn18C complex was chosen. Because it is poorly immunogenic when formulated with AlPO ₄ , since it induces low IgG titers and not all mice respond to it. When formulated with MPL or QS-21, higher IgG titers and greater frequency of responders may be achieved.

AlPO_Four100 μg of MPL ™ or 20 μg of QS-21 ™ was the best adjuvant in this study for Pn18C response. What
Because they induced the highest frequency of responders to this serotype.
You. Nevertheless, IL-12 is not a carrier protein in mice immunized with this complex.
Protein CRM₁₉₇Had a significant effect on the IgG response to In addition, the effect of this cytokine is_FourWas modified by the presence of IL-12 is AlPO_FourSpecifically acts as an adjuvant for vaccines that are formulated without, and increases the dose-dependent increase in IgG titers after primary and secondary vaccination.
Provoked. IL-12 is CRM₁₉₇Enhances the IgG2a response to, which contradicts its ability to favor the induction of TH-1 like helper cells (IFN-γ producers)
do not do. However, IL-12 does not respond to CRM after primary and secondary vaccination. ₁₉₇ The IgG1 response to was also enhanced. IgG1 antibodies are usually associated with TH-2-like helper cells that produce IL-4.

Inclusion of 0.1 μg IL-12 in an AlPO ₄ -based Pn18C conjugate vaccine (which by itself induced a 10-fold higher CRM ₁₉₇ response) had no effect on IgG1 However, IgG2a titers were essentially increased. 0
. The IgG2a titers achieved with 1 μg of IL-12 were as high as those obtained with at least 5 μg of IL-12 in the absence of AlPO ₄ . However, the presence of AlPO ₄ should be noted that you do not exclude enhanced IgG1 response by IL-12. In mice immunized with Pn14 conjugate on AlPO _4, IL-12 of 0.2μg dose enhanced the titer of IgG1, IgG2a Contact and IgG2b for CRM _197. Difference in effect on IgG1 is not may reflect the two immunogenic difference of the complex of the I gG response CRM _197; Pn14 conjugate of AlPO on ₄ enhances the IgG1 response IL- induced IgG titers lower CRM ₁₉₇ than 10 times as room exists for 12, but if were immunized However mice Pn18C complexes on AlPO ₄ did not. The fact that MPL ™ and QS-21 ™ significantly increased IgG1 titers in mice immunized with the Pn18C complex on AlPO ₄ was due to the fact that IgG
One response was not maximally stimulated. Alternatively, the nature of the sugar on the complex may be a factor. In both experiments, higher doses of IL-1
2, the effect was not observed in the absence of AlPO _4, i.e. it resulted in a significant decrease in IgG1, IgG2a and IgG2b titers to CRM _197.

IL-12 probably exerts its adjuvant effect unlike MPL ™ or QS-21 ™. IL-12 was significantly enhanced IgG2a titers CRM ₁₉₇ in mice immunized with Pn18C complexes, but had a minimal effect on the IgG2 b. In contrast, MPL ™ and QS-21 (
TM) enhanced the titers of both IgG subclasses. Dissociation of these two subclasses is induced by cytokines other than or in addition to IFN-γ, which IgG2b drives to switch to IgG2a and is known to mediate the immunomodulatory effects of IL-12. Suggest that. One candidate for driving IgG2b production is TGFb. However, the nature of the antigen cannot be ruled out. Because, in mice immunized with the Pn14 complex, 0.2 μg of IL-12
gG2a and IgG2b were raised to similar levels that were equivalent to the titers promoted by 25 μg of MPL ™.

The studies utilizing bivalent vaccine consisting PnPs14-CRM ₁₉₇ conjugate capsular mixed with a complex of polysaccharides (PnPs6B-CRM ₁₉₇₎ from pneumococcal serotype 6B which is covalently linked to CRM ₁₉₇ Confirmed and extended the findings described above. IL
-12 not only modified the IgG response to the Pn6B complex, but also increased the total IgG titer to this complex. Furthermore, this study relatively low doses of adjuvant activity of IL-12 is further demonstrate more be enhanced to formulate it with AlPO _4. PnPs-14-CRM ₁₉₇ unlike the studies described above using glycoconjugates, IL-12 / AlPO ₄ enhances a subclass of both I gG1 and IgG2a against Pn6B, the apparent enhancement of IgG1 response Pn14 by IL-12 The lack of arguably indicates that it is probably not a generalizable phenomenon. This study further supports the notion that the mechanisms of adjuvant activity by IL-12 and MPL ™ are not equivalent. Both adjuvants increased the IgG1 and IgG2a titers of Pn6B to similar levels, but with MPL (
TM) was more effective at promoting IgG2b and IgG3 antibodies.

IL-12 / AlPO ₄ did not act as an adjuvant for Pn14 IgG response. The reason for this is not clear; however, without intending to be bound by theory, it is most likely that in previous studies, mice were dosed at 1 μg (ie, 10 times more than in Pn6B studies). High) PnPs-14-CRM ₁₉₇ reflects the fact that it was immunized with glycoconjugates. The applicability of IL-12 to more complex pneumococcal vaccines is serotype 1,4,5,6B
, 9V, 14, 18C, 19F and 23F were demonstrated using a 9-valent vaccine containing glycoconjugates from pneumococci. IL-12 was Awa pairs with AlPO _4, in addition to PnPs6B and PnPs14, PnPs4 and PnPs9
Increased IgG2a antibody to V and increased the ability of mice to respond to glycoconjugates prepared with serotype 18C pneumococcal sugar (PnOs-18C-CRM ₁₉₇ ), which is poorly immunogenic in mice .

[0031] In a further example, IL-12 is expressed as a type C meningococcus (Neisseria m.
conjugates (MenC) and type B Hemophilus influenzae (H
bOC) with a glycoconjugate vaccine. 50 ng of the vaccine
Of IL-12 and AlPO ₄ to comprise be formulated is said to have enhanced IgG2a titers against the capsular polysaccharide of MenC, not for HbOC.

The data presented herein show that AlPO ₄ can greatly enhance the efficacy of IL-12 such that essentially lower doses of cytokines can be used. One mechanism capable binds IL-12 is the AlPO _4, thereby is to increase its persistence in the animal; additional studies show that binding to the rapidly alum IL-12 (data Is not shown). Alternatively, local inflammatory effect of AlPO ₄ are, might induce cytokine that enhance the biological activity of IL- 12.

In addition to understanding the physical interaction of IL-12 with AlPO ₄ , several other issues arise from this study using pneumococcal vaccine formulated with IL-12. When AlPO ₄ Considering that enhance the activity of IL-12, to assist the IgG response against glycoconjugates of pneumococcal (adjuvant) Minimum dose of cytokines required to, as well as the IL-5 producing T cells It would be useful to know if activated by glycoconjugate vaccines containing IL-12. These two questions were addressed in the Balb / c mouse study described in Example 4.

The following examples are provided for the purpose of illustrating the invention and should not be construed as limiting the scope of the invention. The teachings of all references cited herein are hereby incorporated by reference. Example Example 1: against the Swiss Webusu coater (Swiss Webster) IgG responses in mice against pneumonia sphere capsular polysaccharide serotypes 14 conjugated to CRM ₁₉₇ on aluminum phosphate (PnPs-14-CRM / AlPO 4) Effect of IL-12 Study Design Swiss Webster mice (1 per group)
0) with 1 μg of PnPs− formulated with 100 μg of AlPO ₄ and without IL-12, with either 0.2 μg, 1 μg or 5 μg of IL-12.
Two immunizations (week 0 and 3) with 14-CRM ₁₉₇ were performed. All vaccines included 0.25% normal mouse serum for the purpose of stabilizing IL-12 when used at low concentrations. PnPs14-CRM ₁₉₇ is a complex of capsular polysaccharide from serotype 14 pneumococci covalently linked to the diphtheria toxin CRM ₁₉₇ , which has been genetically detoxified by reductive amination. Another group received 25 μg instead of IL-12.
MPL ™ (3-O-deacylated monophosphoryl lipid A, RIBI Immunochem Research Inc.)
rch, Inc. ), Hamilton, Montana). Vaccination is 3
They were given subcutaneously apart a week. Serum was collected at week 3 (primary response) and at weeks 5 and 7 (secondary response 2 and 4 weeks after boost). Serum was used for PnPs-
14 were analyzed for IgG antibodies.

Serum was also analyzed for its ability to promote opsonophagocytic killing of type 14 pneumococci by human polynuclear leukocytes (PMN). Type 14 pneumococci were opsonized with dilutions of antiserum and C8-depleted serum as a source of complement. They were then incubated with human polymorphonuclear leukocytes (PMN) and the percentage of surviving bacteria was determined by colony counting. Results Table 1 shows that 1 μg and 5 μg of IL-12 essentially suppressed the anti-PnPs-14 IgG response in mice immunized with the conjugate formulated with AlPO ₄ . The lowest dose (0.2 μg) of the cytokine had no effect on the total IgG response, but resulted in significant changes in the levels of individual immunoglobulin subclasses. Weeks 5 and 7 (2 and 4 weeks after boost, respectively)
In addition, 0.2 μg of IL-12 induced essentially higher IgG2a, IgG2b and IgG3 titers, but IgG1 levels remained essentially unchanged. The characteristics of the IgG subclass induced by 0.2 μg IL-12 are 2
Indistinguishable from that obtained with 5 μg of MPL ™, and the sera from mice receiving these adjuvants had higher opsonin than mice immunized with the vaccine containing only AlPO _4. It had phagocytic activity (Table 2).

Higher doses of IL-12 significantly reduced IgG1 antibodies; with 5 μg of cytokine, IgG1 titers were at least 10-fold lower in mice immunized without IL-12. This effect was evident both during the primary response and after the boost. Increasing the dose of IL-12 is equivalent to IgG2a, IgG
It did not cause a further increase in 2b and IgG3, and, like IgG1, they also decreased, but to varying degrees. IgG2b is
Maximum suppression was shown, as vaccines containing 1 μg or 5 μg of IL-12 induced the same IgG2b titers as those without adjuvant. IgG
2a and IgG3 are less sensitive to the effects of high IL-12 doses;
Even with 5 μg of IL-12, these subclasses were higher than in controls after the second vaccination.

[0037] These studies have shown that IL-12 is obtained by adjusting the response of IgG subclasses against PnPs14- CRM ₁₉₇ conjugate vaccine formulated comprise AlPO _4. A 0.2 μg dose of IL-12 increased the IgG2a, IgG2b and IgG3 responses to Pn14 without affecting the IgG1 response.
Higher doses of IL-12 resulted in a marked decrease in IgG1 and IgG2b titers. IgG2a and IgG3 titers also appeared to decrease at these doses, but they were still higher than in mice immunized in the absence of IL-12. Example 2 shows that changes in the IgG subclass indicate that IFN-γ producing C
Demonstrating that it was associated with enhanced induction of _RM197- specific T cells and a marked decrease in antigen-specific IL-5 production, indicating that the T helper cell phenotype TH-2
It suggests a change to -1 like.

[0038]

[Table 1]

[0039]

[Table 2]

Example 2: Pneumococcal conjugate vaccine formulated with IL-12 (PnPs-
Properties of T-helper cells induced by 14-CRM ₁₉₇ / AlPO ₄ ) Study design A group of 8 Balb / c mice was treated with 1 μg of PnPs- formulated with 100 μg of AlPO ₄ and various doses of IL-12. They were immunized subcutaneously at the base of the tail with 14-CRM ₁₉₇ conjugate. Normal mouse serum (0.25%) was included as carrier protein. One week later, draining lymph node cell suspensions were prepared from half of each group of mice and cultured for 6 days in CRM ₁₉₇ , lysozyme, ConA or medium alone. Culture supernatants from parallel cultures were harvested on days 3 and 6, and ELISA for IFN-γ, IL-5 and IL-10
Assay A.

At three weeks, the remaining mice were bled and re-immunized with the same vaccine formulation used in the first immunization. Mice were bled once more 14 days after the second immunization (Week 5). After 4 days, they were harvested and their draining lymph node cells and spleen cells and, CRM _197, lysozyme, with ConA, or cultured for 6 days in medium alone in. Culture supernatants from parallel cultures were harvested on days 3 and 6 and assayed for IFN-γ, IL-5 and IL-10 by ELISA. Results The PnPs-14-CRM ₁₉₇ / AlPO ₄ vaccine was administered at lower doses of IL-12.
(0.2 μg and 1.0 μg) containing Pn14 at week 5
Greatly enhanced the IgG2a and IgG3 responses to but not IgG1 (see Table 3). Some differences were noted in the results obtained with Balb / c mice and in previous experiments with Swiss Webster.
In this experiment, IL-12 did not dramatically increase the IgG2b antibody to Pn14, and a dose of 5 μg of IL-12 did not increase the IgG1 relative to the control group without cytokine. It did not cause a dramatic (> 10-fold) drop in titer.

[0042] One week after the immunization, lymph node cells from mice immunized without the use of IL-12 is, IFN-γ when stimulated with CRM ₁₉₇ in vitro, IL-1 and IL -10 was produced (Table 4). Adding IL-12 to the vaccine can
Dramatically increased antigen-specific production of FN-γ, and increased IL-5 and IL-10
Abolished the ability of lymphoid cells to produce. Maximal IFN-γ production was obtained with the lowest dose of IL-12 (0.2 μg); higher doses (especially 5 μg) appeared to reduce levels of this cytokine. This was most clearly seen in cultures stimulated with CRM ₁₉₇ in 1 [mu] g / mL. Inhibition of IFN-γ at higher doses of IL-12 may not reflect the generalized phenomenon of inhibition. This is because IFN-γ production in response to ConA was identical regardless of the dose of IL-12 in the vaccine.

Two weeks after the second immunization, lymph node cells and splenocytes from mice immunized with the vaccine containing IL-12 were compared to mice immunized without IL-12. And continued to produce elevated levels of IFN-γ in response to stimulation with CRM ₁₉₇ (Table 5). 0.2 μg as observed 7 days after primary vaccination
To 1.0 μg of IL-12 is sufficient to enhance the IFN-γ response.
Was the optimal application amount. In contrast, however, IL-5 and IL-10 production were differentially affected. 1.0 and 5.0 μg doses of IL-12
The -5 response was essentially eliminated, but by comparison had only a minor effect on IL-10 production. IL-12 (5.0 μg) abolished the ability of splenocytes to produce IL-10, but not the lymph node cells (Tables 5 and 6).

[0044]

[Table 3]

[0045]

[Table 4]

[0046]

[Table 5]

[0047]

[Table 6]

Example 3: Adjuvant activity of IL-12 with poorly immunogenic pneumococcal complex Study Design Swiss Webster mice (1 per group)
0) were immunized with 1 μg of the Pn18C conjugate formulated with or without 100 μg of AlPO ₄ . The vaccine is IL-12 (0.2, 1 or 5μ).
g), supplemented with either 100 μg MPL ™ or 20 μg QS-21 ™. Normal mouse serum (0.5% final) was diluted with IL-12
Was used to stabilize and was added to all vaccines regardless of composition.
Three weeks later, mice were bled and boosted with the same vaccine formulation used in the primary immunization. Bleeds were also collected at weeks 5 and 7 of the study (2 and 4 weeks after boost, respectively). Pooled sera at week 5
It was tested for total IgG and IgG subclasses of Pn18C and CRM _197. To determine the frequency of responders to Pn18C, the serum of individual mice was
Dilutions were made 1:00 and tested by ELISA for IgG antibodies to Pn18C. Results are reported as optical densities. Results The IgG response of Pn18C is presented in Table 7. Addition of IL-12 to the conjugate vaccine formulated with alum had no consistent effect on the IgG response to Pn18C. A 5 μg dose of IL-12 is equivalent to the pooled week 5 serum I
While causing a 3-fold increase in gG titers, vaccines formulated with 1 μg of IL-12 did not appear to induce a Pn18C response. Lowest dose of IL-1
2 (0.1 μg) induced the same response as the vaccine formulated with AlPO ₄ without IL-12. Vaccines formulated with MPL ™ / AlPO ₄ elicited the highest frequency response; 7/10 mice had QS-21 ™ / AlPO ₄
PO ₄ and AlPO ₄ alone (each of which induced 4/10 responders) resulted in an OD> 0.2. Mice immunized with a vaccine containing IL-12 and AlPO ₄ induced 2/10, 0/10 and 1/10 responders at IL-12 doses of 0.1 μg, 1.0 μg and 5 μg, respectively. .

In this experiment, MPL ™ and QS-21 ™ were Pn18C IgG
It caused at most a 3- to 4-fold increase in response. In the absence of AlPO _4, IL-12 had no profound adjuvant effect for IgG responses Pn18C. A vaccine containing a 1 μg dose of IL-12 induced the same Pn18C response as a vaccine without IL-12. Lower and higher doses of I
The vaccine containing L-12 appeared to induce a lower response than the control vaccine. Neither MPL ™ nor QS-21 ™ appeared to enhance the Pn18C IgG response. Among vaccines formulated without AlPO ₄ , QS-21 ™ induced the most frequent responders (7/10 with OD> 0.2), while all other formulations At most 4/10 responders were induced.

To confirm that IL-12 in the vaccine was indeed active, the IgG response of CRM _{197 in} these mice was evaluated. Tables 8 and 9, primary (3 weeks) and secondary post vaccination (week 5), IL-12 is, IgG of CRM ₁₉₇ in mice immunized with vaccines formulated without the AlPO ₄ It is shown to cause a dose-dependent increase in response. In addition, IgG1 and I
IL-12 dose-dependent increase in both titers of gG2a, and IgG at week 5
There was a 2b increase. Week 1 IgG1 and IgG2a titers were similar to those induced by the vaccine formulated with 100 μg MPL ™. In contrast, IgG-2b titers promoted by IL-12 were 20-fold lower than those induced by MPL ™. These data suggest that IgG2a and IgG2b are regulated by different mechanisms, and that IgG2a
IgG2b is dependent on the mechanism activated by IL-12 and IL-2
Controlled by an independent mechanism. These data clearly indicate that IL-12 may act as an adjuvant for IgG responses to protein antigens. Furthermore, increased titers of both IgG1 and IgG2a at least within this model, IL-12 is both PnOs18C-C RM ₁₉₇ TH-1 -like by complex and TH-2 like in the absence of AlPO ₄ Helper Suggests to increase cell priming.

When added to a Pn18C conjugate vaccine formulated with AlPO ₄ , a 0.1 μg dose of IL-12 resulted in a slight, if any, increase in total IgG response to CRM _{197 at} week 3. Caused a 3-fold increase at week 5. However, this dose of IL-12 increased IgG2a titers at week 5 and promoted titers similar to those induced by vaccines containing MPL or QS-21. IL-12 did not significantly increase IgG2b titers. As seen in previous experiments, higher doses of IL-12 resulted in a sharp decrease in IgG titers, with all subclasses affected.

[0052]

[Table 7]

[0053]

[Table 8]

[0054]

[Table 9]

Example 4: Swiss Webster against a bivalent vaccine containing PnPs6B-CRM ₁₉₇ and PnPs-14-CRM ₁₉₇
Effect of IL-12 on IgG Responses in Mice Study Design Swiss Webster mice were challenged with 0.1 μg of PnPs6B-CRM ₁₉₇ glycoconjugate per dose (from serotype 6B pneumococci covalently linked to CRM _197). Capsular polysaccharide complex) and 0.1 μg P per dose
Immunizations were given subcutaneously at weeks 0 and 3 with a vaccine comprising nPs14-CRM ₁₉₇ glycoconjugate. Vaccines were administered with 0, 8, 40 or 200 ng of IL-12, either alone or in combination with 100 μg of alum (AlPO ₄ ). Normal mouse serum (0.25%) was included as a carrier protein to stabilize low concentrations of IL-12. Control mice were immunized with a vaccine formulated with 100 μg of monophosphoryl lipid A (MPL ™). Mice were bled at week 3 (primary response) and at week 5 (secondary response). Serum,
IgG antibodies against Pn6B and Pn14 capsular polysaccharides were tested by ELISA. Results Response to PnPs6B Complex Table 10 illustrates the IgG response of pooled sera to the Pn6B component of the bivalent vaccine. Responses to Pn6B the vaccine properly almost the third week when formulated with non or AlPO ₄ alone contain adjuvants was detected. The highest titer after a single vaccination appeared to be induced by a vaccine containing either MPL ™ or 8-40 ng of IL-12 co-formulated with alum. . These titres, however, were low (ie, less than 3,000). Response week 5 shows that after boosting, the vaccine formulated with 40ng of IL-12 and AlPO ₄ or MPL (TM) induced the highest IgG titers to Pn6B. In the absence of alum, IL-12 in the dose range of 8-200 ng did not increase IgG titers to Pn6B.

The response of the IgG subclass to Pn6B at week 5 is shown in Table 10. The titers of the individual IgG subclasses were determined using adjuvant-free vaccine or AlP
It was similar in mice immunized with O ₄ with vaccines formulated (without IL-12). Furthermore, formulating the vaccine comprising 8~200ng of IL-12 in the absence of AlPO ₄ did not alter the response of the IgG subclasses. In contrast, IL-12 in these doses, when combined with AlPO _4, resulted in IgG1 and IgG2a titers essentially increased for Pn6 B. These titers were similar to those obtained with the vaccine formulated with MPL ™. IL-12 also has IgG2b and IgG3 titers are induction was also increased by the vaccine formulated with AlPO _4; those however, these titers induced by the vaccine formulated with MPL (TM) Seemed to be essentially lower.

[0057] IL-12 and AlPO_FourTo determine if the increase obtained with the combination of was statistically significant, the Pn6B Ig of individual mice in the selected group was
G titers were measured. The geometric mean titer (GMT) is presented in Table 11. This data
Without adjuvant or AlPO_FourThe group immunized with the vaccine formulated alone had similar GMT against Pn6B. AlPO _Four Formulating the vaccine with and 40 ng of IL-12 also has a 29-fold increase in titer over that induced by the vaccine without adjuvant.
I did it. ANOVA (Analysis of variance by JMP software; S
AS Institute, North Carolina
Statistical significance was not found when tested by Cary. data
In comparison of a subset of ANOVA, vaccines containing no adjuvant
Week 5 Responses Induced by AlPO and AlPO_FourAnd statistically significant differences when comparing vaccines formulated with various doses of IL-12.
Of these, AlPO_FourAnd a vaccine formulated with 40 ng of IL-12 had significantly higher Pn than a vaccine formulated without adjuvant.
6B titers were induced. As a further indication of the enhanced immunogenicity of the formulation,
7 of the 10 mice in the group without adjuvant or AlPO_Four1 and 2 animals, respectively, in the group vaccinated with the complex formulated alone
Pn6 greater than or equal to 50,000 compared to mice alone
It had a B titer.

[0058]

[Table 10]

[0059]

[Table 11]

Response to PnPs14 Complex Table 12 shows the IgG response to the PnPs14 component of the vaccine. This data shows that IL-12 in a dose range of 8-40 ng, either alone or when formulated with AlPO ₄ , was found to produce PnPs1 following primary or secondary vaccination.
4 shows no increase in response to 4. Further, subclass analysis indicates that IL-
12 showed no increase in IgG2a titer when formulated with IL-12. In this study, MPL ™ did not have the significant adjuvant effect on the PnPs14 response observed when assaying at least pooled sera in previous studies. Individual sera were assayed at 300-fold dilutions for Pn14 IgG antibodies to gain a concept of the degree of variability of each group's response. The results presented in Table 13 show that there was a large range of responses in each group, ie, except for the group immunized with a vaccine containing MPL ™ whose coefficient of variation (CV) was 0.051, Suggests that CV ranged from 0.229 to 0.587. Thus, MPL ™ might have acted as an adjuvant to the IgG response of Pn14 and might have reduced inter-mouse variability, but IL-12 did not.

[0061]

[Table 12]

[0062]

[Table 13]

Example 5: Monovalent PnPs14-CRM₁₉₇Comparison of the effect of IL-12 in the presence or absence of alum on the immune response of mice to the conjugate vaccine Study Design BALB / c mice (8 per group) were treated with 100 μg of AlPO_FourWith or without IL-12 or as high as 8, 40, 200, 1,000
Or 1 μg of P formulated either with 5,000 ng of IL-12
nPs14-CRM₁₉₇The conjugate was immunized subcutaneously at week 0. Normal mouse serum (0.25%) was used as a carrier protein for stabilizing low concentrations of IL-12.
Included. In the first week, a lymph node cell suspension was prepared from half the mice in each group,
And evaluated in vitro for antigen-specific cytokine production. Those spleen
Was also harvested and weighed. In the third week, the remaining mice are bled and
Reimmunization was performed with the same vaccine formulation used in the first vaccination. In the fifth week,
Mice immunized twice were bled, their spleens weighed and their splenocytes
Were evaluated for cytokine production. PnPs14 and CRM₁₉₇IgG and IgG subclass titers were measured in pooled sera. Assays individually
When performed using sera from different mice, the results were expressed as geometric mean titers (GMT).
Expressed as Results Effect of IL-12 on spleen weight one week after immunization One week after the first immunization, AlPO_FourMice that received 5,000 ng of IL-12 (but not the lower dose of IL-12) in the absence of
Significantly greater than those receiving vaccines containing neither IL-12
Although having spleen weight (Table 14). AlPO_FourVaccine induces greater spleen weight when formulated with 40-5000 ng of IL-12
Was. Paired comparisons were performed with 200 or 1000 ng of IL-12 and AlPO. _Four The vaccine prescribed with AlPO_FourSame dose of IL-12 in the absence of
Showed that they induced greater spleen weight than those prescribed with. as a whole
, This data is_FourPrescribing IL-12 along with it causes the biological activity of the cytokine, ie, increased spleen weight one week after vaccination.
Indicates that he greatly enhanced his ability to rub. Effect of IL-12 on IgG response to PnPs14 First, pooled sera were assayed for IgG antibodies to PnPs14.
(Table 15). The clearest indication of the adjuvant effect is AlPO_FourAnd after primary immunization with a vaccine containing 8 to 40 ng of IL-12
. This combination is AlPO_FourHad a 17- to 21-fold increase in IgG titers for mice immunized with vaccines formulated without either IL-12 or IL-12.
I did. AlPO_FourThe combination of and IL-12 produces a greater response than when used individually;_FourAnd a dose of 40 ng of IL-12 caused a 4-fold and 5-fold increase in IgG titers at week 3, respectively. Al
PO_FourAnalysis of individual sera from mice immunized with a vaccine containing (Table 16) showed that 8 ng of IL-12 showed AlPO after primary vaccination._FourInduces PnPs14 IgG titers 5 times higher than adjuvanted vaccine
It was shown that. This difference in titers was statistically significant. Higher doses of I
L-12 did not enhance the response. 1,000-5,000 ng dose of IL
-12 caused a significant decrease in the IgG titer of PnPs14. Second immunity
After cropping, only 40 ng of IL-12 was dosed with AlPO_FourCaused a significant increase (3-fold) in the PnPs14 titer induced by the Vaccine-based vaccine.

The data from pooled sera suggest that the combination of AlPO ₄ and 8-40 ng of IL-12 increased IgG1 titers after primary immunization. After two vaccinations, IL-12 was pooled (Table 15) and individual sera (Table 1).
As shown by analysis of 7), it did not enhance the IgG1 titers against PnPs14 in mice immunized with the complex in the absence of AlPO _4. Furthermore, AlP
O ₄ in between the mice immunized with vaccines containing, addition of 8 to 200ng of IL-12 did not result in higher IgG1 titers after two vaccinations (Table 17).

The most dramatic effect of IL-12 was to essentially increase the IgG2a response of PnPs14 at week 5. This vaccine was observed in both cases formulated without the case or AlPO ₄ contained AlPO ₄ (Table 18). In the absence of A LPO _4, a statistically significant increase in GMT of IgG2a (14 a stone 42 times) it was obtained in IL-12 of 1,000ng to no 8. Similarly, 8
Although １，1,000 ng of IL-12 enhanced the ability of AlPO ₄ -containing vaccines to induce IgG2a antibodies, 8 and 40 ng doses of IL-1 were used in this study.
Only the titer induced by 2 was statistically higher. Overall, the best I
gG2a titers were induced by multiple coalesced formulated with IL-12 of AlPO ₄ and 40 ng. This differs significantly from IgG2a titers induced by IL-12 of 40ng absence of AlPO _4, again, the adjuvant activity of IL-12 showed that enhanced by alum.

[0066] IgG2b and IgG3 titers were assayed with pooled sera only (Table 15). 8 to the dose of IL-12 in the range of 1,000ng is, if it is Moni co-formulated with an AlPO _4, has been promoted essential increase in IgG3 titers after primary and secondary immunization, but It did not in the absence of AlPO _4. No consistent effect of IL-12 on IgG2b titers was shown. Effect of IL-12 on IgG response to CRM _{197 The} IgG response to CRM ₁₉₇ was also evaluated to see if there was a difference between the effect of IL-12 on the protein carrier versus the polysaccharide portion of the conjugate (
Table 19). In the absence of AlPO _4, IL-12 of 40ng seemed to increase moderately the IgG titers to CRM ₁₉₇ after two vaccinations. While only the highest IgG titers against CRM _197, the vaccine was obtained when formulated with IL-12 both AlPO ₄ and 8～40Ng. Enhanced adjuvant activity of AlPO ₄ and IL-12 which are both co-formulation, by itself, 40
Although ng of IL-12 and AlPO ₄ resulted in 6 fold and 17-fold increase in IgG titers in week 5, however, when combined together as shown by the finding that increased was 147 times . IL-12, the vaccine regardless of whether formulated without including it if it were formulated containing the AlPO _4, enhanced the I gG1 response to CRM ₁₉₇ (Tables 19 and 20). IL-12 was essentially increases the fifth week of IgG2a titers to CRM ₁₉₇ after immunization with the vaccine you containing AlPO ₄ (Table 19). Again, the optimal dose of IL-12 appeared to be 40 ng. This cytokine appeared to increase the IgG2b titers induced by vaccine containing AlPO _4. CRM ₁₉₇ specific T cell cytokine IL-12 effect a second vaccination 2 weeks after for the feature of cytokine production by spleen cells harvested (week 5) is, IFN-gamma-producing cells and IL-5 production The effect of IL-12 on priming of both cells was shown. Splenocytes from immunized mice in the absence of AlPO ₄ and IL-12 has been produced IL-5 in detectable levels when stimulated with CRM ₁₉₇ in vitro, but IFN-gamma chopsticks No (Table 21
). Formulating the vaccine with IL-12 appears to enhance the induction of IL-5 producing cells, with peak activity occurring at 40 ng of cytokine. Higher doses of IL-12 result in reduced production of IL-5, in effect cytokines are produced by mice immunized with a conjugate vaccine containing 1,000 to 5,000 ng of IL-12 Did not. To be convinced of IFN-γ production,
It was only detected in splenocytes of mice immunized with the vaccine formulated with 5,000 ng of IL-12. Where the vaccine was formulated comprise AlPO _4, and one that brought priming addition of IL-12 in 8n g was produced IFN-gamma copious amounts cells, in the absence of this cytokine, antigen-specific IL- Only 5 productions were detected. Priming for maximum IFN-γ production is between 40 and 1,
It appears to occur at 000 ng of IL-12. Addition of 5,000 ng of IL-12 abolished the ability of the vaccine to prime for IL-5 producing cells.

[0067]

[Table 14]

[0068]

[Table 15]

[0069]

[Table 16]

[0070]

[Table 17]

[0071]

[Table 18]

[0072]

[Table 19]

[0073]

[Table 20]

[0074]

[Table 21]

Example 6: IL- on humoral response to a 9-valent pneumococcal glycoconjugate vaccine
12 / AlPO_FourEffect of IL-12 Study Design Evaluation of the effect of IL-12 on IgG response to pneumococcal glycoconjugate vaccine
Titers were determined from serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F.
Expanded to a nine-valent vaccine composed. Swiss Webster (Swiss W
ebster) Mice were challenged with 0.1, 1 or 5 μg of vaccine at 0 and 3 weeks.
Immunized with tin (carbohydrate weight). The vaccine was isolated, AlPO_Four(100 μg) or mixed with 50, 200 or 1,000 ng of IL-12
Combined AlPO_FourTogether with the drug. Normal mouse serum was not included in this vaccine. Serotypes 4, 6B, 9V, 14, 18C and carrier protein CRM ₁₉₇ IgG response to was evaluated by ELISA at week 5 (ie, 2 weeks after booster challenge). Results CRM at Week 5₁₉₇Response to AlPO_FourThe addition of IL-12 to a vaccine containing₁₉₇I for
This resulted in a dose-dependent increase in gG2a and IgG2b antibodies. This is tested
The conjugate was found at all doses (Table 22). The increased IgG2a titer is 5
It is evident in mice receiving 0 ng of cytokine, and also 1,000 ng of cytokine.
Was the largest. This contrasts with other studies, where the maximum IgG2a potency was
With 40-100 ng of cytokine added to the alum-based vaccine
And higher doses of IL-12 resulted in a reduced immune response. Laboratory
The reason for the difference in dose response between studies is unknown. It is the difference between vaccines,
Previous studies to stabilize multivalent versus monovalent or low concentrations of cytokines
It may be related to omitting the normal mouse serum included in the vaccine.
Response to pneumococcal polysaccharide AlPO_FourFormulating a nine-valent vaccine containing PnPs4, PnPs6B, PnPs9V, especially when using the lowest dose of conjugate (0.1 μg)
And enhanced IgG responses to several serotypes including PnPs14 (
Tables 24-27). Addition of IL-12 further enhances the IgG response to these serotypes.
Did not seem to increase. However, in the case of a PnPs18C response, Al
PO_FourAddition of 50 or 1,000 ng of IL-12 to 5 μg of vaccine containing, a higher geometric mean IgG titer for this serotype, and a
Results in a higher proportion of mice with IgG titers of PnPs18C above 00
(Table 23). Responses to PnPs1, 5, 19F and 23F were not evaluated
won.

Addition of IL-12 to a 9-valent vaccine containing AlPO ₄ resulted in a dose-dependent increase in IgG2a titers for PnPs4, PnPs6B, PnPs9V and PnPs14 (Tables 24-27). In general, the increase in IgG2a is CR
Parallel to that of the _M197 response, the highest titers were obtained with 1,000 ng of IL-12. In contrast to experiments using a monovalent PnPs14 conjugate or a bivalent PnPs6B / PnPs14 vaccine, a dose of 50 ng IL-12 had little or no effect on the IgG2a response to these serotypes. The exception is the IgG2a response to PnPs14. This dose of cytokine appeared to enhance the response to this serotype (Table 27).

Overall, this study shows that IL-12 can enhance the response of the complement-binding IgG2a antibody subclass to multiple pneumococcal serotypes present in multivalent vaccines.

[0078]

[Table 22]

[0079]

[Table 23]

[0080]

[Table 24]

[0081]

[Table 25]

[0082]

[Table 26]

[0083]

[Table 27]

Example 7: IL-12 and A on the immune response to Neisseria meningitidis type C (menC) glycoconjugate vaccine
Effect Study Design This study LPO ₄ evaluated the IL-12 with a vaccine against N. meningitidis (Neiserria meningitidis) Type C (MenC). Swiss Webster (Swiss Webster) mice, to a 0 and 3 weeks, alone, AlPO ₄ (100 [mu] g), or MenC of IL-12 (50ng) and AlP O combination of ₄ with prescribed the 0.1μg or 1μg Immunized with glycoconjugates. Normal mouse serum was not added to the vaccine. Mice were bled at weeks 3 and 5 and sera were analyzed by ELISA for IgG antibodies to the polysaccharide of menC. Results When immunized with higher doses of the conjugate, equivalent menC IgG titers were generated regardless of adjuvant formulation. However, IL-12 / A to vaccines
The addition of LPO ₄ was formulated with AlPO ₄ (but not IL-12),
Alternatively, it resulted in higher IgG2a titers to the polysaccharide than without adjuvant.

In mice immunized with lower doses of the conjugate, higher meningC titers were obtained when the vaccine was formulated with AlPO ₄ (Table 28). Addition of IL-12 to the adjuvant did not increase total IgG titers, but did not increase IgG2 titers.
a resulted in> 10-fold increase in antibody. These data, IL-12 together with AlPO ₄ indicates that there is a possibility of promoting the induction of complement binding IgG subclasses against menC glycoconjugate vaccine.

[0086]

[Table 28]

Example 8: Hemophilus influenzae
Type b Effect Study Design This study of IL-12 and AlPO ₄ for immune response against glycoconjugate vaccine (HbOC) is Haemophilus influenzae (Hemophilus influenzae
) IL-12 was evaluated using a vaccine against type b. Swiss Webster mice (10 per group) were challenged at weeks 0 and 3 with a complex saccharide consisting of capsular polysaccharide (HibPs) from Hemophilus influenzae type b conjugated to CRM _197. Immunized with 0.1 μg or 1.0 μg of the quality vaccine. Vaccine (HbOC), was administered alone or AlPO ₄ (100 [mu] g) or IL-12 (50ng) jointly with the mixture of our and AlPO _4. Normal mouse serum was not added to the vaccine. Mice were bled at 3 and 5 weeks. Antibody response to HibPs was measured using a Farr assay that measures total antibody binding to sugars regardless of isotype, ie, IgM, IgG and IgA. The response of the IgG subclass was measured by ELISA. In addition, the response of IgG and IgG subclass to CRM ₁₉₇ was also measured by ELISA. Results The titer (primary response) of the anti-HibPs antibody in the pooled sera from the third week blood draws was independent of AlPO ₄ or IL-12, regardless of the dose of the conjugate used for immunization. and they did not differ between immunized mice with the vaccine formulated with AlPO ₄ (Table 29). Analysis of pooled sera from week 5 blood draws shows that in mice immunized with 1 μg HbOC containing IL-12 and alum,
It suggested that it resulted in at least 10-fold higher anti-HibPs than when given with alum or without adjuvant (Table 30). However, analysis of individual mouse sera showed that this was due to a single mouse with a titer of approximately 10,000 μg / mL. When expressing the results as geometric mean titers, there was no evidence of an enhanced HibPs response by IL-12. The response of the IgG subclass to HibPs was determined by ELISA in pooled sera.
It was evaluated by SA. The combination of IL-12 and AlPO ₄ appeared to increased 3 fold IgG2a titers in mice immunized with multiple coalescence 1 [mu] g. However, this was not different from the titer obtained with a vaccine assisted by AlPO ₄ alone. In mice immunized with 0.1 μg HbOC, IL-12 and A
lPO ₄ did not increase IgG2a titers against HibPs. Ig that adjuvant combinations of IL-12 / A lPO ₄ were active is shown by analysis of anti-CRM ₁₉₇ response (Table 31), where that is increased relative to a carrier protein
G2a titers were seen in mice immunized with either dose of the conjugate.

[0088]

[Table 29]

[0089]

[Table 30]

[0090]

[Table 31]

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

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF terms (reference) 4C085 AA03 AA05 AA38 BA14 BA16 CC07 FF02 FF18

Claims (56)

[Claims] 1. An antigen of pneumococcus, an adjuvant amount of interleukin-12
And a mixture of minerals in suspension, and optionally a physiologically acceptable vehicle. 2. The vaccine composition according to claim 1, wherein the interleukin-12 is adsorbed on the mineral suspension. 3. The vaccine composition according to claim 1, wherein the interleukin-12 is human interleukin-12. 4. The vaccine composition according to claim 1, wherein the mineral in suspension is an aqueous suspension of alum. 5. The vaccine composition according to claim 4, wherein the alum is aluminum hydroxide or aluminum phosphate. 6. The pneumococcal antigen, wherein the pneumococcal capsular polysaccharide serotypes 1, 4, 5, and 6.
The vaccine composition of claim 1, wherein the vaccine composition is selected from the group consisting of 6B, 9V, 14, 18C, 19F and 23F, and combinations thereof. 7. The vaccine composition of claim 1, wherein the pneumococcal antigen is conjugated to a carrier molecule. 8. The vaccine composition according to claim 7, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof. 9. The vaccine composition according to claim 8, wherein the carrier molecule is CRM ₁₉₇ . 10. An antigen of pneumococcus and an adjuvant amount of interleukin-1.
And administering to a mammalian host an effective amount of a vaccine composition, optionally comprising a mixture of minerals in suspension and optionally a physiologically acceptable vehicle. A method of eliciting an immune response to an antigen. 11. The method of claim 10, wherein the interleukin-12 is adsorbed on the mineral suspension. 12. The method according to claim 10, wherein the interleukin-12 is human interleukin-12. 13. The method according to claim 10, wherein the mineral in suspension is an aqueous suspension of alum. 14. The method according to claim 13, wherein the alum is aluminum hydroxide or aluminum phosphate. 15. The pneumococcal antigen is a serotype 1, 4, 5 of the pneumococcal capsular polysaccharide.
11. The method of claim 10, wherein the method is selected from the group consisting of, 6B, 9V, 14, 18C, 19F and 23F, and combinations thereof. 16. The method of claim 10, wherein the pneumococcal antigen is conjugated to a carrier molecule. 17. The carrier molecule of claim 1, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin, and non-toxic variants thereof.
7. The method according to 6. 18. The method according to claim 17, wherein the carrier molecule is CRM ₁₉₇ . 19. Pneumococcal antigen, adjuvant amount of interleukin-1
A pneumococcal vaccine comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of minerals and a suspension of minerals, and optionally comprising a physiologically acceptable vehicle. A method for increasing the IFN-γ response to 20. An antigen of pneumococcus and an adjuvant amount of interleukin-1
A pathogen comprising administering to a mammalian host an effective amount of an immunogenic composition comprising a mixture of minerals and a suspension-like mineral, and optionally comprising a physiologically acceptable vehicle. A method for deriving a complement fixation antibody for a protective response to 21. Pneumococcal antigen, adjuvant amount of interleukin-1
An immunogenic composition comprising a mixture of the minerals in a suspension and a suspension, and optionally comprising a physiologically acceptable vehicle. 22. The immunogenic composition of claim 21, wherein the interleukin-12 is adsorbed on the mineral suspension. 23. The immunogenic composition according to claim 21, wherein the interleukin-12 is human interleukin-12. 24. The immunogenic composition according to claim 21, wherein the mineral in suspension is an aqueous suspension of alum. 25. The immunogenic composition according to claim 24, wherein the alum is aluminum hydroxide or aluminum phosphate. 26. The pneumococcal antigen is a serotype 1, 4, 5 of the pneumococcal capsular polysaccharide.
22. The immunogenic composition of claim 21, wherein the immunogenic composition is selected from the group consisting of, 6B, 9V, 14, 18C, 19F and 23F, and combinations thereof. 27. The immunogenic composition of claim 21, wherein the pneumococcal antigen is conjugated to a carrier molecule. 28. The carrier molecule of claim 2, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.
8. The immunogenic composition according to 7. 29. The immunogenic composition according to claim 28, wherein the carrier molecule is CRM ₁₉₇ . 30. An antigen of meningococcus, an adjuvant amount of interleukin-1
A vaccine composition comprising a mixture of minerals and a suspension, and optionally comprising a physiologically acceptable vehicle. 31. The vaccine composition of claim 30, wherein the interleukin-12 is adsorbed on the mineral suspension. 32. The vaccine composition of claim 30, wherein the interleukin-12 is human interleukin-12. 33. The vaccine composition of claim 30, wherein the mineral in suspension is an aqueous suspension of alum. 34. The vaccine composition according to claim 33, wherein the alum is aluminum hydroxide or aluminum phosphate. 35. The method according to claim 35, wherein the antigen of the meningococcal is Neisseria meli.
31. The vaccine composition according to claim 30, which is a type C capsular polysaccharide of Ningitidis). 36. The vaccine composition of claim 30, wherein the meningococcal antigen is conjugated to a carrier molecule. 37. The carrier molecule of claim 3, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.
7. The vaccine composition according to 6. 38. The vaccine composition of claim 37, wherein the carrier molecule is CRM ₁₉₇ . 39. An antigen of meningococcus, an adjuvant amount of interleukin-1
Meningitis comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of minerals and a suspension of minerals, and optionally comprising a physiologically acceptable vehicle. A method of eliciting an immune response to a bacterial antigen. 40. The method of claim 39, wherein the interleukin-12 is adsorbed on the mineral suspension. 41. The method of claim 39, wherein the interleukin-12 is human interleukin-12. 42. The method of claim 39, wherein the mineral in suspension is an aqueous suspension of alum. 43. The method of claim 42, wherein the alum is aluminum hydroxide or aluminum phosphate. 44. A method wherein the antigen of meningococci is Neisseria meli.
40. The method of claim 39 which is a type C capsular polysaccharide of Ningitidis). 45. The method of claim 39, wherein the meningococcal antigen is conjugated to a carrier molecule. 46. The carrier molecule of claim 4, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin, and non-toxic variants thereof.
5. The method according to 5. 47. The method of claim 46, wherein the carrier molecule is CRM ₁₉₇ . 48. An antigen of meningococcus, an adjuvant amount of interleukin-1
An immunogenic composition comprising a mixture of the minerals in a suspension and a suspension, and optionally comprising a physiologically acceptable vehicle. 49. The immunogenic composition of claim 48, wherein the interleukin-12 is adsorbed on the mineral suspension. 50. The immunogenic composition according to claim 48, wherein the interleukin-12 is human interleukin-12. 51. The immunogenic composition according to claim 48, wherein the mineral in suspension is an aqueous suspension of alum. 52. The immunogenic composition according to claim 51, wherein the alum is aluminum hydroxide or aluminum phosphate. 53. A method wherein the antigen of Neisseria meningitidis is Neisseria melis.
49. The immunogenic composition of claim 48, which is a type C capsular polysaccharide of N. nigitidis). 54. The immunogenic composition of claim 48, wherein the meningococcal antigen is conjugated to a carrier molecule. 55. The carrier molecule according to claim 5, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.
5. The immunogenic composition according to 4. 56. The immunogenic composition according to claim 55, wherein the carrier molecule is CRM ₁₉₇ .