WO1999040936A2 - Pneumococcal and meningococcal vaccines formulated with interleukin-12 - Google Patents

Pneumococcal and meningococcal vaccines formulated with interleukin-12 Download PDF

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WO1999040936A2
WO1999040936A2 PCT/US1999/002847 US9902847W WO9940936A2 WO 1999040936 A2 WO1999040936 A2 WO 1999040936A2 US 9902847 W US9902847 W US 9902847W WO 9940936 A2 WO9940936 A2 WO 9940936A2
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il
composition according
interleukin
vaccine
suspension
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PCT/US1999/002847
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WO1999040936A3 (en
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Vincent J. Laposta
John H. Eldridge
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American Cyanamid Company
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/095Neisseria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55522Cytokines; Lymphokines; Interferons
    • A61K2039/55527Interleukins
    • A61K2039/55538IL-12
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
    • A61K2039/6037Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Abstract

This invention pertains to vaccine compositions comprising a mixture of antigen, such as a pneumococcal or meningococcal antigen, and interleukin IL-12, which may be adsorbed onto a mineral in suspension. The pneumococcal or meningococcal antigen may be conjugated to a carrier molecule. These vaccine compositions modulate the protective immune response to the antigen.

Description

PNEUMOCOCCAL AND MENINGOCOCCAL VACCINES FORMULATED WITH INTERLEUKIN-12

BACKGROUND OF THE INVENTION

The immune system uses many mechanisms for attacking pathogens; however, not all of these mechanisms are necessarily activated after immunization. Protective immunity induced by vaccination is dependent on the capacity of the vaccine to elicit the 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 on the basis of the cytokines they produce, and that the distinct cytokine profile observed in these cells determines their function. This T cell model includes two major subsets: TH-1 cells that produce IL- 2 and interferon γ (IFN-γ) which augment both cellular and humoral immune responses, and TH-2 cells that produce IL-4, IL-5 and IL-10 which augment humoral immune responses (Mosmann et al . , J. Immunol . 126: 234,8 (1986)). It is often desirable to enhance the immunogenic potency of an antigen in order to obtain a stronger immune response in the organism being immunized and to strengthen host resistance to the antigen-bearing agent. A substance that enhances the immunogenicity of an antigen with which it is administered is known as an adjuvant. For example, certain lymphokines have been shown to have adjuvant activity, thereby enhancing the immune response to an antigen (Nencioni et al . , J. Immunol . 139:800-804 (1987); EP285441 to Howard et al . ) .

SUMMARY OF THE INVENTION This invention pertains to vaccine compositions comprising a mixture of one or more pneumococcal or meningococcal antigens, the interleukin IL-12 and a mineral in suspension. The IL-12 can be either adsorbed onto the mineral suspension or simply mixed therewith. In a particular embodiment of the invention, the IL-12 is adsorbed onto a mineral suspension such as alum (e.g. , aluminum hydroxide or aluminum phosphate) . These vaccine compositions modulate the protective immune response to the antigen; that is, the vaccine composition is capable of quantitatively and qualitatively improving the vaccinated host's antibody response, and quantitatively increasing cell-mediated immunity for a protective response to a pathogen. In a particular embodiment of the invention, the antigen is a pneumococcal or meningococcal antigen; the antigens are optionally conjugated to a carrier molecule, such as in a pneumococcal or meningococcal glycoconjugate .

Studies described herein show that IL-12 can modify the humoral response of mice immunized with pneumococcal and meningococcal glycoconjugate vaccines formulated with aluminum phosphate (A1P04) The particular pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F, (Pnl, Pn4, Pn5, Pn6B, Pn9V, Pnl4, Pnl8C, Pnl9F, Pn23F) , and the meningococcal polysaccharide is type C (Men C) . These serotypes, however, are not to be construed to limit the scope of the invention, as other pneumococcal and meningococcal serotypes are also suitable for use herein. Moreover, it will be apparent to the skilled artisan that conjugation to a carrier molecule, such as the CRM197 protein exemplified herein, is optional, depending upon the immunogenicity of the selected pneumococcal or meningococcal antigen. Doses of IL-12 ranging from about 8 ng to about

1,000 ng increased the IgGl, IgG2a, IgG2b and IgG3 response to alum-adsorbed Pnl4 or PnβB. In addition they increased the IgG2a response to Pn4 and Pn9V. Doses of IL-12 of about 5,000 ng markedly reduced the overall IgG titers to Pnl4, and especially the IgGl and IgG2b titers.

The invention also pertains to methods for preparing an immunogenic composition or a vaccine composition comprising a mixture of antigen and IL-12 with a mineral in suspension. In particular, the IL-12 is adsorbed onto the mineral suspension. The invention also pertains to methods for eliciting or increasing a vaccinee ' s IFN-γ-producing T cells and complement-fixing IgG antibodies for a protective immune response, comprising administering to a mammalian, e.g., human or primate, host an effective amount of a vaccine composition comprising a mixture of antigen, IL-12 and a mineral in suspension in a physiologically acceptable solution. In particular, the IL-12 is adsorbed onto the mineral suspension. DETAILED DESCRIPTION OF THE INVENTION

Work described herein reveals the ability of IL-12 to increase the immune response to alum-based pneumococcal vaccines, particularly serotype 14 and serotype 6B pneumococcal glycoconjugate vaccines, and meningococcal vaccines, particularly type C, to increase the proportion of complement-fixing IgG2a and IgG2b antibodies. As described herein, PnPs-14-CRM197 vaccine comprises a serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant of diphtheria toxoid

(cross-reacting material) designated CRM197, and PnPsβB- CRM197 vaccine comprises a serotype 6B pneumococcal polysaccharide conjugated to CRM197. IL-12 was compared to MPL® (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamitton, Montana), which in mice is a potent adjuvant for pneumococcal vaccines. In a separate experiment conducted in Balb/c mice, the effect of IL-12 on the cytokine profile of the CRM- specific T cells induced by the exemplary conjugate vaccines on alum was examined.

IL-12 is produced by a variety of antigen- presenting cells, principally macrophages and monocytes. It is a critical element in the induction of TH-1 cells from naive T cells. Production of IL-12 or the ability to respond to it has been shown to be critical in the development of protective TH-1-like responses, for example, during parasitic infections, most notably Leishmaniasis (Scott et al . , U.S. Patent No. 5,571,515). The effects of IL-12 are mediated by IFN-γ produced by NK cells and T helper cells. Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor, is a heterodimeric cytokine (Kobayashi et al . , J. Exp . Med. 170 : 827 (1989)). The 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 reveal the utility of IL-12 as an adjuvant in a pneumococcal or meningococcal vaccine, and particularly a pneumococcal or meningococcal glycoconjugate vaccine. Accordingly, this invention pertains to vaccine compositions comprising a mixture of such an antigen, IL-12 and a mineral in suspension. In a particular embodiment of the invention, the IL-12 is adsorbed onto a mineral suspension such as alum (e.g., aluminum hydroxide or aluminum phosphate) . These vaccine compositions modulate the protective immune response to the antigen; that is, the vaccine composition is capable of eliciting the vaccinated host's complement-fixing antibodies for a protective response to the pathogen. In a particular embodiment of the invention, the antigen is a pneumococcal antigen, particularly a pneumococcal polysaccharide; the pneumococcal antigen is optionally conjugated to a carrier molecule, such as in a pneumococcal glycoconjugate. The particular pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F; however, these serotypes are not to be construed to limit the scope of the invention, as other serotypes are also suitable for use herein. In another embodiment of the invention, the antigen is a meningococcal antigen, particularly a meningococcal polysaccharide; the meningococcal antigen is optionally conjugated to a carrier molecule, such as in a meningococcal glycoconjugate. Type C Neisseria meningi tidis is exemplified herein; however, this type is not to be construed to limit the scope of the invention, as other types are also suitable for use herein. IL-12 can be obtained from several suitable sources . It can be produced by recombinant DNA methodology; for example, the gene encoding human IL-12 has been cloned and expressed in host systems, permitting the production of large quantities of pure human IL-12. Also useful in the present invention are biologically active subunits or fragments of IL-12. Commercial sources of recombinant human and murine IL-12 include Genetics Institute, Inc. (Cambridge, MA) .

The antigen of this invention, e.g., a pneumococcal or meningococcal antigen or a pneumococcal or meningococcal glycoconjugate, can be used to elicit an immune response to an antigen in a mammalian host. For example, the antigen can be a serotype 14 or 6B pneumococcal polysaccharide or a portion thereof which retains the ability to stimulate an immune response. Additional suitable antigens include polysaccharides from other encapsulated bacteria and conjugates thereof, secreted toxins and outer membrane proteins .

The method comprises administering to the mammal, e.g., human or primate, an immunologically effective dose of a vaccine composition comprising a mixture of an antigen, such as a pneumococcal antigen or a pneumococcal conjugate, and an adjuvant amount of IL-12 adsorbed onto a mineral in suspension. As used herein, an "immunologically effective" dose of the vaccine composition is a dose which is suitable to elicit an immune response. The particular dosage of IL-12 and the antigen will depend upon the age, weight and medical condition of the mammal to be treated, as well as on the method of administration. Suitable doses will be readily determined by the skilled artisan. The vaccine composition can be optionally administered in a pharmaceutically or physiologically acceptable vehicle, such as physiological saline or ethanol polyols such as glycerol or propylene glycol .

The vaccine composition may optionally comprise additional adjuvants such as vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamin, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N,N- dicoctadecyl-N' -N'bis (2-hydroxyethyl-propane diamine) , methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dime hylglycine, tuftsin; immune stimulating complexes; oil emulsions; liposaccharides such as MPL® and mineral gels. The antigens of this invention can also be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly- lactide-co-glycolides , or ISCOMS (immunostimulating complexes) , and supplementary active ingredients may also be employed. The antigens of the present invention can also be administered in combination with bacterial toxins and their attenuated derivatives . The antigens of the present invention can also be administered in combination with other lymphokines, including, but not limited to, IL-2, IL-3 , IL-15, IFN-γ and GM-CSF.

The vaccines can be administered to a human or animal by a variety of routes, including but not limited to parenteral, intradermal, transdermal (such as by the use of slow release polymers) , intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration. The amount of antigen employed in such vaccines will vary depending upon the identity of the antigen. Adjustment and manipulation of established dosage ranges 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, in particular, humans. Typically, the IL-12 and the antigen will be co-administered; however, in some instances the skilled artisan will appreciate that the IL-12 can be administered close in time but prior to or after vaccination with the antigen.

The pneumococcal and meningococcal antigens of the present invention can be coupled to a carrier molecule in order to modulate or enhance the immune response. Suitable carrier proteins include bacterial toxins rendered safe by chemical or genetic means for administration to mammals and immunologically effective as carriers. Examples include pertussis, diphtheria, and tetanus toxoids and non- oxic mutant proteins (cross-reacting materials (CRM) ) , such as the non-toxic variant of diphtheria toxoid, CRM197. Fragments of the native toxins or toxoids, which contain at least one T- cell epitope, are also useful as carriers for antigens, as are outer membrane protein complexes. Methods for preparing conjugates of pneumococcal antigens and carrier molecules are well known in the art and can be found, for example, in Dick and Burret, Contrib Microbiol Immunol . 10:48-114 (Cruse M, Lewis RE Jr, eds; Basel, Krager (1989)) and U.S. Patent No. 5,360,897 (Anderson eϋ al . ) .

The adjuvant action of IL-12 has a number of important implications. The adjuvanticity 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 can enhance the ability of antigens which are weakly antigenic or poorly immunogenic to elicit an immune response. It may also provide for safer vaccination when the antigen is toxic at the concentration normally required for effective immunization. By reducing the amount of antigen, the risk of toxic reaction is reduced.

Typically, vaccination regimens call for the administration of antigen over a period of weeks or months in order to stimulate a "protective" immune response. A protective immune response is an immune response sufficient to protect the immunized organism from productive infection by a particular pathogen or pathogens to which the vaccine is directed. As shown in the Examples, in an alum-formulated vaccine, comprising IL-12 adsorbed onto AlP04 and a serotype 14 or serotype 6B pneumococcal polysaccharide conjugated to CRM197, which normally induces a response dominated by IgGl, 0.2 μg of IL-12 substantially increased the IgG2a and IgG3 subclasses in both Balb/c and Swiss Webster mice, but had little or no effect on IgGl. Enhancement of IgG2b to Pnl4 was seen with Swiss Webster mice; 0.2 μg of IL-12 had the same effect as 25 μg of MPL® on the IgG subclass response to Pnl4, suggesting that IL-12 is at least 100-fold more biologically active than MPL® in this regard. As expected from the IgG subclass distribution, especially the enhanced IgG2a response, the opsonophagocytic activity of the antisera for Pnl4 pneumococci from mice receiving 0.2 μg IL-12 was higher than that of controls and was equivalent to that of mice immunized with vaccine formulated with a much larger amount of MPL®.

Briefly, IgG2a and IgG2b antibodies are very efficient at activating the complement system, whereas IgGl antibodies are not. The complement system consists of a series of plasma proteins which come together around IgG2a or IgG2b bound to antigen (e.g., bacteria) to form a large molecular complex. Deposition of this complex on the surface of bacteria results in the killing of the bacteria by perforating the cell membrane (bactericidal activity) or by facilitating the recognition of the bacteria by phagocytic cells (such as polymorphonuclear cells (PMN) used in this study) , which take up the bacteria and kill them (opsonophagocytosis) . Increasing the dose of IL-12 profoundly reduced the

IgGl and IgG2b responses. The reduction in these immunoglobulin subclasses was not simply due to a change in the kinetics of the antibody response, as has been observed in the hen egg lysozyme (HEL) system (Buchanan, Van Cleave and Metzger, Abstract #1945; 9th

International Congress of Immunology (1995)), as these subclasses were reduced at all time points tested. The effect on IgGl was expected given that switching of B cells to this subclass requires IL-4, a TH-2 cytokine whose production is inhibited by IL-12. The reduction in IgG2b, however, was not expected since in previous studies increased levels of IgG2b have 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 regulation of IgG2b. For example, Germann et al . (Eur J. Immunol 25:823-829 (1995)) found that treating mice with anti-IFN-γ inhibited the ability of IL-12 to promote IgG2a responses, but not IgG2b. Other studies have implicated TGF-β 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, it is possible that high doses of IL-12 may affect TGF-β production or responsiveness to it. IFN-γ is critical for the induction of IgG2a antibodies to T-dependent protein antigens (Finkelman and Holmes, Annu . Rev. Immunol . S:303-33 (1990)) and IgG3 responses to T-independent antigens (Snapper et al . , J. Exp . Med. 175:1367-1371 (1992)). Increased IFN- γ response was consistently found after a single vaccination with vaccine (PnPs-14-CRM197) containing IL- 12 and A1P04 and after boosting. The effect of IL-12 on the TH-2 cytokines IL-5 and IL-10 appears to depend on when the lymphoid cells are harvested after vaccination, and possibly on the particular cytokine. Exogenous IL- 12 completely abolished antigen-specific IL-5 and IL-10 production by lymph node cells (LNC) harvested 1 week after primary vaccination. After secondary vaccination, differences were seen between these two cytokines; IL-5 production by either LNC or splenocytes was completely abolished by 1 μg IL-12 in the vaccine, but IL-10 production was largely unaffected after boosting. It is unclear whether these differences are due to setting up the cultures at different times or reflect the expansion of a TH-2-like population upon subsequent revaccination. The latter possibility is consistent with data from Wolf and colleagues (Bliss et al . , J. Immunol 155:887-894 (1996) ) , indicating that IL-4-producing T cells can be recovered from Balb/c mice previously immunized with vaccine containing IL-12 and boosted with soluble antigen. In their studies, IL-4 was detected even if IL-12 was included in the secondary vaccine. The presence of TH-2 cytokines after boosting may explain why, in Balb/c mice, even high levels of IL-12 could not reduce the secondary IgGl response to below control levels (conjugate vaccine on alum) . Unlike the Balb/c mice, high doses of IL-12 severely inhibited the IgGl response of Swiss Webster mice. Whether this is associated with decreased production of TH-2 cytokines after the second vaccination is unclear. In the present studies, IL-12 exhibited either only immunomodulatory activity or behaved both as a "classical" adjuvant, and a immunomodulator, depending on the vaccine. In the study with PnPsl4-CRM197 the IgG response (especially the primary response) to the vaccine was not substantially elevated by the presence of the cytokine but certain subclasses, i.e. IgG2a and IgG3 , were elevated whereas the others were unchanged or diminished. Thus, IL-12 is useful for altering the humoral response to an already immunogenic vaccine. It is possible that in these studies the adjuvant activity of IL-12 was masked by the presence of alum, which is an adequate adjuvant on its own for the highly immunogenic PnPs-14 conjugate. The adjuvanticity of IL-12 may be better demonstrated in the absence of alum, by reducing the dose of conjugate or by using a poorly immunogenic conjugate. Thus, further evaluations were carried out using IL-12 in the presence and absence of alum with PnPs6B conjugate vaccines, which are less immunogenic in Swiss Webster mice than PnPs-14 conjugate vaccines. An additional study was designed to address the issue of IL-12 adjuvant activity for a poorly immunogenic pneumococcal conjugate. The Pnl8C conjugate was chosen, as it is poorly immunogenic when formulated with A1P04, i.e., it induces low IgG titers and not all mice respond to it. When formulated with MPL or QS-21, higher IgG titers and a greater frequency of responders can be achieved.

One hundred μg MPL® plus A1P04 or 20 μg QS-21™ were the best adjuvants in this study for a Pnl8C response as they induced the highest frequency of responders to this serotype. Nonetheless, IL-12 had marked effects on the IgG response to the carrier protein, CRM197, in mice immunized with this conjugate. Moreover, the effects of the cytokine were modified by the presence of A1P04 in the vaccine. IL-12 clearly acted as an adjuvant for vaccines formulated without A1P04, causing a dose- dependent increase in IgG titers after primary and secondary vaccination. IL-12 enhanced the IgG2a response to CRM197, which is consistent with its ability to favor the induction of TH-1-like helper cells (IFN-γ producers) . However, IL-12 also enhanced the IgGl response to CRM197 after primary and secondary vaccination. IgGl antibodies are normally associated with TH-2-like helper cells which produce IL-4. Inclusion of 0.1 μg IL-12 into an AlP04-based Pnl8C conjugate vaccine (which on its own induced a 10-fold higher CRM197 response) had no effect on IgGl but substantially increased the IgG2a titer. The IgG2a titer achieved with 0.1 μg IL-12 was at least as high as that obtained with 5 μg IL-12 in the absence of A1P04. It should be noted, however, that the presence of A1P04 does not preclude the enhancement of IgGl responses by IL-12. In mice immunized with the Pnl4 conjugate on A1P04, a 0.2 μg dose of IL-12 enhanced the IgGl, IgG2a and IgG2b titers to CRM197. The differences in the effect on IgGl may reflect differences in the immunogenicity of the two conjugates for CRM197 IgG responses; the Pnl4 conjugate on A1P04 induced 10-fold lower CRM197 IgG titers so that there was room for IL-12 to enhance an IgGl response, but not when mice were immunized with Pnl8C conjugate on AlP04. The fact that MPL® and QS-21™ markedly increased the IgGl titers in mice immunized with Pnl8C conjugate on A1P04 indicates that the IgGl response had not been maximally stimulated. Alternatively, the nature of the saccharides on the conjugates may be a factor. In both experiments, higher doses of IL-12 resulted in a marked diminution of the IgGl, IgG2a and IgG2b titers to CRM197, an effect that was not seen in the absence of A1P04. IL-12 probably exerts its adjuvant effect differently than MPL® or QS-21™. IL-12 markedly enhanced the CRM197 IgG2a titers in mice immunized with Pnl8C conjugate but had minimal effects on IgG2b. In contrast, MPL® and QS-21™ enhanced the titers of both IgG subclasses. The dissociation of these two subclasses suggests that IgG2b is induced by cytokines other than, or in addition to, the IFN-γ that drives switching to IgG2a and is known to mediate the immunomodulatory effects of IL-12. One candidate for driving IgG2b production is TGFb. The nature of the antigen cannot be excluded, however, since in mice immunized with Pnl4 conjugate, 0.2 μg IL-12 caused IgG2a and IgG2b to be elevated to similar levels which were equivalent to the titers promoted by 25 μg MPL®. Studies utilizing a bivalent vaccine consisting of a PnPsl4-CRM197 conjugate mixed with a conjugate of capsular polysaccharide from serotype 6B pneumococci covalently linked to CRM197 (PnPs6B-CRM197) confirmed and extended the above-described findings. IL-12 not only modified the IgG response to the Pn6B conjugate, but also enhanced the overall IgG titer to the conjugate. Moreover, this work further demonstrates that the adjuvant activity of relatively low doses of IL-12 is enhanced by formulating it with A1P04. Unlike the above- described studies with PnPs-14-CRM197 glycoconjugate, IL- 12 /A1P04 enhanced both the IgGl and IgG2a subclasses to Pn6B, indicating that the apparent lack of enhancement of the Pnl4 IgGl response by IL-12 is probably not a generalizable phenomenon. This work further supports the idea that the mechanisms of adjuvant activity by IL- 12 and MPL® are not equivalent. Both adjuvants enhanced the Pn6B IgGl and IgG2a titers to similar levels, but MPL® was more effective at promoting IgG2b and IgG3 antibodies.

IL-12/AlP04 did not act as an adjuvant for the Pnl4 IgG response. The reason for this is not clear; however, without wishing to be bound by theory, this most likely reflects the fact that in previous studies mice were immunized with a 1 μg dose of PnPs-14-CRM197 glycoconjugate, i.e., 10-fold higher than in the Pn6B studies . The applicability of IL-12 to more complex pneumococcal vaccines was demonstrated using a nonavalent vaccine containing glycoconjugates from serotype 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F pneumococci. The combination of IL-12 with A1P04 enhanced the IgG2a antibodies to PnPs4 and PnPs9V, in addition to PnPs6B and PnPsl4, and increased the ability of mice to respond to glycoconjugate prepared with serotype 18C pneumococcal saccharide (PnOs-18C-CRM197) which is poorly immunogenic in mice.

In further examples, IL-12 was tested with a glycoconjugate vaccine against type C Neiserria meningitidis (MenC) and a glycoconjugate vaccine against type B Hemophilus influenzae (HbOC) . Formulating that vaccine with 50 ng IL-12 and A1P04 enhanced the IgG2a titers to MenC capsular polysaccharide although not to HbOC.

The data presented herein indicate that A1P04 can greatly enhance the potency of IL-12 so that substantially lower doses of the cytokine can be used. One possible mechanism is that IL-12 binds to AlP04, thereby enhancing its persistence in the animal; additional studies indicate that IL-12 rapidly binds to alum (data not shown) . Alternatively, the local inflammatory effect of A1P04 may induce cytokines that potentiate the biological activity of IL-12.

In addition to understanding the physical interaction of IL-12 with A1P04, several other issues arise from the present work with pneumococcal vaccines formulated with IL-12. Given that A1P04 enhances the activity of IL-12, it would be useful to know the minimal dose of cytokine needed to adjuvant the IgG response to pneumococcal glycoconjugates , as well as whether IL-5-producing T cells are activated by IL-12- containing glycoconjugate vaccines. These two questions were addressed in the studies in Balb/c mice described in Example 4.

The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incorporated herein by reference .

EXAMPLES EXAMPLE 1: Effect of IL-12 on the IgG response of

Swiss Webster mice to serotype 14 pneumococcal capsular polysaccharide conjugated to CRM197 on aluminum phosphate (PnPs-14-CRM/AlP04)

STUDY DESIGN

Swiss Webster mice (10 per group) were immunized twice (at weeks 0 and 3) with 1 μg PnPs-14-CRM197 formulated with 100 μg AlP04 and either no IL-12, 0.2 μg , 1 μg or 5 μg IL-12. All vaccines included 0.25% normal mouse serum for the purpose of stabilizing the IL-12 when used at low concentrations. PnPsl4-CRM197 is a conjugate of capsular polysaccharide from serotype 14 pneumococci covalently linked to the genetically detoxified diphtheria toxin, CRM197, by reductive amination. Another group received 25 μg MPL® (3-0- deacylated monophosphoryl lipid A, RIBI Immunochem Research, Inc., Hamilton Montana) instead of IL-12. The vaccinations were given subcutaneously three weeks apart . Sera were collected at week 3 (primary response) and weeks 5 and 7 (secondary responses 2 and 4 weeks after boosting) . The sera were analyzed for IgG antibodies to PnPs-14. The sera were also analyzed for the ability to promote opsonophagocytic killing of type-14 pneumococci by human polymorphonuclear cells (PMN) . Type 14 pneumococci were opsonized with dilutions of antisera and C8-depleted serum as a source of complement. They were then incubated with human polymorphonuclear cells (PMN) , and the percent of bacteria surviving was determined by colony counts .

RESULTS

Table 1 shows that 1 μg and 5 μg IL-12 substantially reduced the anti-PnPs-14 IgG response in mice immunized with conjugate formulated with AlP04. The lowest dose (0.2 μg) of cytokine had no effect on the total IgG response but caused major changes in the levels of the individual immunoglobulin subclasses. At weeks 5 and 7 (2 and 4 weeks after boosting, respectively), 0.2 μg IL-12 induced substantially higher IgG2a, IgG2b and IgG3 titers but left the IgGl levels essentially unaltered. The IgG subclass profile induced by 0.2 μg IL-12 was indistinguishable from that obtained with 25 μg MPL®, and sera from mice receiving these adjuvants had higher opsonophagocytic activity than those from mice immunized with a vaccine containing only A1P04 (Table 2) . The higher doses of IL-12 markedly reduced the IgGl antibodies; at 5 μg cytokine, IgGl titers were at least 10-fold lower than in mice immunized without IL-12. This effect was apparent both during the primary response and after boosting. Increasing the IL-12 dose did not cause further increases in IgG2a, IgG2b and IgG3 , and, like IgGl, they also declined, although to varying degrees. IgG2b showed the greatest reduction such that vaccines containing 1 μg or 5 μg IL-12 induced the same IgG2b titer as those without adjuvant. IgG2a and IgG3 were less sensitive to the effects of high IL- 12 dose; even with 5 μg IL-12, after the second vaccination these subclasses were higher than in the controls . These studies showed that IL-12 could modulate the

IgG subclass response to a PnPsl4-CRM197 conjugate vaccine formulated with A1P04. A 0.2 μg dose of IL-12 increased the IgG2a, IgG2b and IgG3 response to Pnl4 without affecting the IgGl response. Higher doses of IL-12 resulted in a marked reduction in the IgGl and IgG2b titers. IgG2a and IgG3 titers also appeared to decline at these doses, but they were still higher than in mice immunized in the absence of IL-12. Example 2 demonstrates that the IgG subclass changes were associated with enhanced induction of IFN-γ-producing, CRM197-specific T cells and a marked reduction in antigen-specific IL-5 production, suggesting a change in the T helper cell phenotype from TH-2-like to TH-1-like. Table 1: Effect of IL-12 on the immunogenicity of PnPs- 14-CRM197/alum vaccine

Figure imgf000023_0001

Table 2 : Opsonophagocytic activity of sera of mice immunized with PnPs-14-CRM197/AlP04 formulated with IL-12 or MPL®

Figure imgf000024_0001

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EXAMPLE 2 : Nature of T helper cells induced by

Pneumococcal conjugate vaccine (PnPs-14- CRM197/A1P04) formulated with IL-12

STUDY DESIGN Groups of eight (8) Balb/c mice were immunized subcutaneously at the base of the tail with 1 μg PnPs- 14-CRM197 conjugate formulated with 100 μg A1P04 and different doses of IL-12. Normal mouse serum (0.25%) was included as a carrier protein. One week later, draining lymph node cell suspensions were prepared from half the mice in each group and cultured with CRM197, lysozyme, ConA or in medium alone for 6 days. Culture supernatants from parallel cultures were harvested at day 3 and day 6 and assayed for IFN-γ, IL-5 and IL-10 by ELISA.

At three weeks, the remaining mice were bled and reimmunized with the same vaccine formulation used in the first immunization. Fourteen days after the second immunization (week 5), the mice were bled once more. Four days later their draining lymph node cells and splenocytes were harvested and cultured for six days with CRM197, lysozyme, ConA or in medium alone. Culture supernatants from parallel cultures were harvested at day 3 and day 6 and assayed for IFN-γ, IL-5 and IL-10 by ELISA.

RESULTS

Formulating PnPs-14-CRM197/AlP04 vaccine with the lower doses of IL-12 (0.2 μg and 1.0 μg) greatly enhanced the IgG2a and IgG3 responses to Pnl4 at week 5, but not IgGl (see Table 3) . Several differences were seen between the results obtained with Balb/c mice and Swiss Webster mice in the previous experiment; in this experiment IL-12 did not dramatically increase the IgG2b antibodies to Pnl4, nor did the 5 μg IL-12 dose cause the dramatic (> 10-fold) reduction in IgGl titers relative to the control group without cytokine.

One week after immunization, lymph node cells from mice immunized without IL-12 produced IFN-γ, IL-5 and IL-10 when stimulated with CRM197 in vi tro (Table 4) . Adding IL-12 to the vaccine dramatically increased the antigen-specific production of IFN-γ and abolished the ability of the lymphoid cells to produce IL-5 and IL-10. Maximal IFN-γ production was obtained with the lowest dose of IL-12 (0.2 μg) ; higher doses, particularly 5 μg, appeared to reduce the levels of this cytokine. This was most clearly seen in cultures stimulated with 1 μg/mL CRM197. The reduction in IFN-γ with higher doses of IL-12 may not reflect a generalized suppressive phenomenon since IFN-γ production in response to Con A was the same 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 vaccine containing IL-12 continued to produce elevated levels of IFN-γ in response to stimulation with CRM197 compared to mice immunized without IL-12 (Table 5) . As observed 7 days after primary vaccination, 0.2 μg to 1.0 μg IL-12 were optimal doses of IL-12 for augmentation of an IFN-γ response. In contrast, however, IL-5 and IL-10 production were differentially affected. The 1.0 and 5.0 μg doses of IL-12 essentially eliminated the IL-5 response but, by comparison, had only a minor effect on IL-10 production. IL-12 (5.0 μg) abolished the ability of splenocytes but not lymph node cells to produce IL-10 (Tables 5 and 6) .

Table 3 : Effect of IL-12 on immune response to an alum- based PnPsl4 glycoconjugate vaccine in Balb/c Mice

Figure imgf000028_0001

Table 4 : Cytokines produced by lymph node cells taken 7 days after single immunization with PnPs-14 conjugate formulated with A1P04 and IL-12

Day 6 Cultures

Antigen No 0.2 μg 1.0 μg 5.0 μg

Cytokine in vi tro μg/ml IL-12 IL-12 IL-12 IL-12

IFN-γ CRM 30 23.2 102.7 60.5 32.2

(U/mL) CRM 1 <0.75 65.2 28.6 8.7

Lysozyme 30 <0.75 2.9 6.6 4.5

Con A 1 43.8 97.1 107.1 105.4

Medium - <0.75 3.6 10.6 5.2

IL-5 CRM 30 7.2 <0.22 <0.22 <0.22

(ng/mL) CRM 1 2.2 <0.22 <0.22 <0.22

Lysozyme 30 <0.22 <0.22 <0.22 <0.22

Con A 1 <0.22 <0.22 <0.22 <0.22

Medium - <0.22 <0.22 <0.22 <0.22

IL-10 CRM 30 10.4 0.8 0.21 0.21

(ng/mL) CRM 1 2.6 0.21 0.21 0.21

Lysozyme 30 <0.14 0.21 0.21 0.21

Con A 1 <0.14 0.21 0.21 0.21

Medium - <0.14 0.21 0.21 0.21

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Table 5 : Cytokine production by splenocytes two weeks after secondary vaccination with PnPs-14 conjugate formulated with A1P04 and IL-12

Day 6 Cultures

Antigen No 0.2 μg 1.0 μg 5.0 μg

Cytokine in vitro μg/ml IL-12 IL-12 IL-12 IL-12

IFN-γ CRM 30 7.0 98.4 83.2 50.9

(U/mL)

CRM 1 1.0 89.2 76.8 16.4

Lysozyme 30 <0.4 <0.4 <0.3 <0.3

Con A 1 42.7 48.7 50.6 49.5

IL-5 CRM 30 13.2 3.1 0.6 <0.2

(ng/mL) CRM 1 4.5 4.4 0.8 <0.2

Lysozyme 30 <0.3 <0.3 <0.2 <0.2

Con A 1 <0.3 <0.3 <0.2 <0.2

IL-10 CRM 30 8.6 4 7.1 0.6

(ng/mL) CRM 1 1.1 2.5 1.7 <0.2

Lysozyme 30 <0.2 <0.2 <0.3 <0.2

Con A 1 0.5 0.4 <0.3 <0.2

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Table 6 : Cytokine production by lymph node cells two weeks after secondary vaccination with PnPs-14 conjugate formulated with A1P04 and IL-12

Day 6 Cultures

Antigen No 0.2 μg 1.0 μg 5.0 μg

Cytokine in vi tro μg/ml IL-12 IL-12 IL-12 IL-12

IFN-γ CRM 30 9.8 86.9 58.7 62.0

(U/mL) CRM 1 0.6 78.6 62.9 36.8

Lysozyme 30 <0.4 <0.4 <0.3 <0.3

Con A 1 17.7 57.6 45.7 69.0

IL-5 CRM 30 12.5 1.4 <0.2 0.5

(ng/mL) CRM 1 4.8 2.2 <0.2 <0.2

Lysozyme 30 <0.3 <0.3 <0.2 <0.2

Con A 1 1.1 <0.3 <0.2 <0.2

IL-10 CRM 30 11.3 9.9 7.2 3.6

(ng/mL) CRM 1 4.4 5.5 3.3 1

Lysozyme 30 <0.2 <0.2 <0.2 <0.2

Con A 1 <0.2 <0.2 <0.2 <0.2

EXAMPLE 3 : IL-12 adjuvant activity with poorly immunogenic Pneumococcal conjugate

Study Design

Swiss-Webster mice (10 per group) were immunized with 1 μg Pnl8C conjugate formulated with or without 100 μg A1P04. The vaccines were supplemented with either IL- 12 (0.2, 1 or 5 μg) , 100 μg MPL® or 20 μg QS-21™. Normal mouse serum (0.5% final) was used to stabilize the diluted IL-12 and was added to all vaccines, regardless of composition. Three weeks later, the mice were bled and boosted with the same vaccine formulation used at the primary immunization. Bleeds were also taken at weeks 5 and 7 of the study (2 and 4 weeks after boosting, respectively) . Pooled sera were tested at week 5 for Pnl8C and CRM197 total IgG and IgG subclasses. To determine the frequency of responders to Pnl8C, the sera for individual mice were diluted 1/500 and tested by ELISA for IgG antibodies to Pnl8C. Results are reported as optical density.

Results

The Pnl8C IgG responses are presented in Table 7. The addition if IL-12 to alum-formulated conjugate vaccine had no consistent effect on the IgG response to Pnl8C. A dose of 5 μg of IL-12 caused a 3 -fold rise in the IgG titer of pooled week 5 sera, whereas vaccine formulated with 1 μg of IL-12 appeared to induce no Pnl8C response. The lowest dose of IL-12 (0.1 μg) induced the same response as the A1P04-formulated vaccine not containing IL-12. The vaccine formulated with MPL®/AlP04 induced the highest frequency of responses; 7/10 mice gave OD>0.2, in contrast to QS-21™/AlP04 and

A1P04 alone, each of which induced 4/10 responders. Mice immunized with vaccine containing IL-12 plus A1P04 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™ caused at most a

3- to 4-fold increase in the PnlδC IgG response. In the absence of A1P04, IL-12 did not have a profound adjuvant effect on the PnlδC IgG response. The vaccine containing a 1 μg dose of IL-12 induced the same Pnl8C response as vaccine without IL-12. Vaccines containing the lower and higher doses of IL-12 appeared to induce lower responses than the control vaccine. Neither MPL® nor QS-21™ appeared to enhance the Pnl8C IgG response. Among the vaccines formulated without AlP0 QS-21™ induced the highest frequency of responders (7/10 with OD>0.2), whereas all other formulations induced 4/10 responders, at most.

To confirm that the IL-12 in the vaccine was indeed active, the CRM197 IgG response in these mice was evaluated. Tables 8 and 9 show that after primary (week 3) and secondary (week 5) vaccination, IL-12 causes a dose-dependent increase in CRM197 IgG response in mice immunized with vaccine formulated without A1P04. Moreover, there was an IL-12 dose-dependent increase in both IgGl and IgG2a titers at weeks 3 and 5, as well as an increase in IgG2b at week 5. The IgGl and IgG2a titers at week 5 were similar to those induced by vaccine formulated with 100 μg MPL®. In contrast, the IgG2b titers promoted by IL-12 were 20-fold lower than those induced by MPL®. These data suggest that IgG2a and IgG2b are controlled by different mechanisms, IgG2a being dependent on a mechanism activated by IL-12 and IgG2b being controlled by an IL-12-independent mechanism. These data clearly indicate that IL-12 can act as adjuvant for IgG responses to a protein antigen. Moreover th