AU759391B2

AU759391B2 - Pneumococcal and meningococcal vaccines formulated with interleukin-12

Info

Publication number: AU759391B2
Application number: AU25965/99A
Authority: AU
Inventors: John H. Eldridge; Vincent J. Laposta
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 1998-02-12
Filing date: 1999-02-10
Publication date: 2003-04-10
Anticipated expiration: 2019-02-10
Also published as: IL137809A; WO1999040936A3; CN1200730C; AU2596599A; BR9907884A; JP2010265279A; WO1999040936A2; JP2010006827A; KR20010040898A; IL137809A0; KR100585408B1; JP2002502882A; CN1292706A; EP1053015A2; CA2320223A1

Description

WO 99/40936 PCTIUS99/02847 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 cellmediated 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 y (IFN-y) 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. 126:2348 (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 SUBSTITUTE SHEET rule 26 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 meningoccal 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 aluminum hydroxide or aluminum phosphate).

These vaccine compositions modulate the protective immune response to the antigen; that to 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.

Thus, according to a first aspect of this invention there is provided a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum and optionally comprising a physiologically acceptable vehicle.

According a second embodiment of this invention there is provided a method of eliciting an immune response to a pneumococcal antigen, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

25 According to a third embodiment of this invention there is provided a method of enhancing IFN-y response to a pneumococcal vaccine, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising, alum and optionally comprising a physiologically acceptable vehicle.

30 According to a fourth embodiment of this invention there is provided a method of eliciting complement-fixing antibodies for a protective response to a pathogen,

I

comprising administering to a mammalian host an effective amount of an immunogenic composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a S31 physiologically acceptable vehicle.

[R:\LIBUU]02496.doc:dxn According to a fifth embodiment of this invention there is provided an immunogenic composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

According to a sixth embodiment of this invention there is provided a vaccine composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

According to a seventh embodiment of this invention there is provided a method of 0o eliciting an immune response to a meningococcal antigen, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

According to a eighth embodiment of this invention there is provided an immunogenic composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension conprising alum, and optionally comprising a physiologically acceptable vehicle.

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 (AIPO 4 The particular pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F, (Pnl, Pn4, Pn6B, Pn9V, Pnl4, Pnl8C, Pnl9F, Pn23F), o o ••go °oo [R:\LIBUU]02496.doc:dxn WO 99/40936 PCT/US99/02847 -3and 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 CRM, 97 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 IgG1, IgG2a, IgG2b and IgG3 response to alum-adsorbed Pnl4 or Pn6B. 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 IgG1 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-y-producing T cells and complement-fixing IgG antibodies for a protective immune response, comprising administering to a mammalian, 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.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCTIUS99/02847 -4- 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-CRM 9 vaccine comprises a serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant of diphtheria toxoid (cross-reacting material) designated CRM 19 7 and PnPs6B-

CRM

97 vaccine comprises a serotype 6B pneumococcal polysaccharide conjugated to CRM 197 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 CRMspecific T cells induced by the exemplary conjugate vaccines on alum was examined.

IL-12 is produced by a variety of antigenpresenting 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-l-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-y produced by NK cells and T helper cells. Interleukin-12 (IL-12), SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 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 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.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -6- 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 meningitidis 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, 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, human or primate, an immunologically effective SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/US99/02847 -7dose 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, hexadecylamin, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N,Ndicoctadecyl-N'-N'bis (2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines, pyran, dextransulfate, poly IC, carbopol; peptides, muramyl dipeptide, dimethylglycine, 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 polylactide-co-glycolides, or ISCOMS (immunostimulating SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -8complexes), 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-y 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 SUBSTITUTE SHEET rule 26) WO 99/40936 PCTIS99/02847 -9rendered safe by chemical or genetic means for administration to mammals and immunologically effective as carriers. Examples include pertussis, diphtheria, and tetanus toxoids and non-toxic mutant proteins (cross-reacting materials such as the non-toxic variant of diphtheria toxoid, CRM 19 Fragments of the native toxins or toxoids, which contain at least one Tcell 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 JM, Lewis RE Jr, eds; Basel, Krager (1989)) and U.S. Patent No. 5,360,897 (Anderson et 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 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 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 AlPO 4 and a serotype 14 or serotype 6B pneumococcal polysaccharide conjugated to CRM 197 which normally induces a response dominated by IgG1, 0.2 ug 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 IgG1. Enhancement of IgG2b to Pnl4 was seen with Swiss Webster mice; 0.2 Ag of IL-12 had the same effect as Mg 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 ug 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 IgG1 antibodies are not. The complement system consists of a series of plasma proteins which come together around IgG2a or IgG2b bound to antigen 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 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -11- (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 IgG1 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-y 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-y inhibited the ability of IL-12 to promote IgG2a responses, but not IgG2b. Other studies have implicated TGF-P 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-P production or responsiveness to it.

IFN-y is critical for the induction of IgG2a antibodies to T-dependent protein antigens (Finkelman SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -12and Holmes, Annu. Rev. Immunol. 8:303-33 (1990)) and IgG3 responses to T-independent antigens (Snapper et al., J. Exp. Med. 175:1367-1371 (1992)). Increased IFNy response was consistently.found after a single vaccination with vaccine (PnPs-14-CRM, 9 containing IL- 12 and AlPO 4 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 production by lymph node cells (LNC) harvested 1 week after primary vaccination. After secondary vaccination, differences were seen between these two cytokines; production by either LNC or splenocytes was completely abolished by 1 ug IL-12 in the vaccine, but 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 156:887-894 (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 SUBSTITUTE SHEET rule 26 WO 99/40936 PCTIUS99/02847 -13mice, high doses of IL-12 severely inhibited the IgG1 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-CRM 97 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 AlPO 4 it induces low IgG titers and not all mice respond to it. When formulated with MPL or QS-21, SUBSTITUTE SHEET rule 26 WO 99/40936 PCT[US99/02847 -14higher IgG titers and a greater frequency of responders can be achieved.

One hundred ig MPL® plus A1PO 4 or 20 pg QS-21 M 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, CRM 197 in mice immunized with this conjugate. Moreover, the effects of the cytokine were modified by the presence of AlPO 4 in the vaccine. IL-12 clearly acted as an adjuvant for vaccines formulated without A1PO 4 causing a dosedependent increase in IgG titers after primary and secondary vaccination. IL-12 enhanced the IgG2a response to CRM 9 which is consistent with its ability to favor the induction of TH-l-like helper cells (IFN-y producers). However, IL-12 also enhanced the IgG1 response to CRM 19 7 after primary and secondary vaccination. IgG1 antibodies are normally associated with TH-2-like helper cells which produce IL-4.

Inclusion of 0.1 ug IL-12 into an AlPO 4 -based Pnl8C conjugate vaccine (which on its own induced a higher CRM 19 response) had no effect on IgG1 but substantially increased the IgG2a titer. The IgG2a titer achieved with 0.1 ig IL-12 was at least as high as that obtained with 5 pg IL-12 in the absence of A1PO 4 It should be noted, however, that the presence of AlPO 4 does not preclude the enhancement of IgG1 responses by IL-12. In mice immunized with the Pnl4 conjugate on A1PO 4 a 0.2 pg dose of IL-12 enhanced the IgG1, IgG2a and IgG2b titers to CRM, 19 The differences in the SUBSTITUTE SHEET rule 26 WO 99/40936 PCT[US99/02847 effect on IgG1 may reflect differences in the immunogenicity of the two conjugates for CRM, 7 IgG responses; the Pnl4 conjugate on AlPO 4 induced lower CRM1 97 IgG titers so that there was room for IL-12 to enhance an IgG1 response, but not when mice were immunized with Pn18C conjugate on A1PO 4 The fact that MPL® and QS-21 T markedly increased the IgG1 titers in mice immunized with Pnl8C conjugate on AlPO indicates that the IgG1 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 IgG1, IgG2a and IgG2b titers to CRM 19 an effect that was not seen in the absence of AlPO 4 IL-12 probably exerts its adjuvant effect differently than MPL® or QS-21

T

IL-12 markedly enhanced the CRM 197 IgG2a titers in mice immunized with Pnl8C conjugate but had minimal effects on IgG2b. In contrast, MPL® and QS-21 T M 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-y 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 ig IL-12 caused IgG2a and IgG2b to be elevated to similar levels which were equivalent to the titers promoted by 25 ug MPL®.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCTIUS99/02847 -16- Studies utilizing a bivalent vaccine consisting of a PnPsl4-CRM 97 conjugate mixed with a conjugate of capsular polysaccharide from serotype 6B pneumococci covalently linked to CRM, 19 (PnPs6B-CRM 197 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 AlPO 4 Unlike the abovedescribed studies with PnPs-14-CRM 19 glycoconjugate, IL- 12/AlPO 4 enhanced both the IgG1 and IgG2a subclasses to Pn6B, indicating that the apparent lack of enhancement of the Pnl4 IgG1 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/A1PO 4 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 pg dose of PnPs-14-CRM 9 glycoconjugate, 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 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -17pneumococci. The combination of IL-12 with AlPO 4 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-CRM 9 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 AlPO enhanced the IgG2a titers to MenC capsular polysaccharide although not to HbOC.

The data presented herein indicate that A1PO 4 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 A1PO 4 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 AlPO 4 may induce cytokines that potentiate the biological activity of IL-12.

In addition to understanding the physical interaction of IL-12 with A1PO 4 several other issues arise from the present work with pneumococcal vaccines formulated with IL-12. Given that AlPO 4 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- SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -18containing 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 CRM9 7 on aluminum phosphate (PnPs-14-CRM/AlPO 4 STUDY DESIGN Swiss Webster mice (10 per group) were immunized twice (at weeks 0 and 3) with 1 kg PnPs-14-CRM 1 formulated with 100 Mg AlPO 4 and either no IL-12, 0.2 yg, 1 Mg or 5 Mg IL-12. All vaccines included 0.25% normal mouse serum for the purpose of stabilizing the IL-12 when used at low concentrations. PnPsl4-CRM 19 7 is a conjugate of capsular polysaccharide from serotype 14 pneumococci covalently linked to the genetically detoxified diphtheria toxin, CRM 197 by reductive amination. Another group received 25 yg MPL® deacylated monophosphoryl lipid A, RIBI Immunochem Research, Inc., Hamilton Montana) instead of IL-12. The vaccinations were given subcutaneously three weeks SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -19apart. 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 mg and 5 4g IL-12 substantially reduced the anti-PnPs-14 IgG response in mice immunized with conjugate formulated with A1PO 4 The lowest dose (0.2 Mg) 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 gg IL-12 induced substantially higher IgG2a, IgG2b and IgG3 titers but left the IgG1 levels essentially unaltered. The IgG subclass profile induced by 0.2 Ag IL-12 was indistinguishable from that obtained with 25 Aug MPL®, and sera from mice receiving these adjuvants had higher opsonophagocytic activity than those from mice immunized with a vaccine containing only A1PO 4 (Table 2).

SUBSTITUTE SHEET rule 26) WO 99/40936 PCTIUS99/02847 The higher doses of IL-12 markedly reduced the IgGl antibodies; at 5 ug cytokine, IgGl titers were at least 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 IgG1, they also declined, although to varying degrees. IgG2b showed the greatest reduction such that vaccines containing 1 jg or 5 ug 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 ig 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-CRM, 97 conjugate vaccine formulated with AlPO 4 A 0.2 pg dose of IL-12 increased the IgG2a, IgG2b and IgG3 response to Pnl4 without affecting the IgG1 response. Higher doses of IL-12 resulted in a marked reduction in the IgG1 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-y-producing,

CRM

197 -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-l-like.

SUBSTITUTE SHEET rule 26 WO 99/40936 PTU9/24 PCTIUS99/02847 -21- Table 1: Effect of IL-12 on the immnunogenicity of PnPs- 14-CRM, 9 ,/alum vaccine ______PnPsl4 IgG Response Time Adjuvant Dose (gsg) XgG IgG1 IgG2a IgG2b IgG3 week 3 None 56,035 8,394 481 298 1,312 IL-12 5 13,137 480 2,417 388 2,39E IL-12 1 26,131 1,5211 3,249 736 3,858 .0.2 90,220 13,7791 4,731. 1,454 7,944 MPL9@ 25 46,451 14,3031 1,506 8,506 18,203 week 5 None 531,270 189,571 5,507 14,463 18,158 IL-12 .5 231,015 16,900 28,719 6,002 56,982 IL-12 1 198,044 36,327 27,420 11,841 30,740 IL-12 0.2 722,360 305,623 60,701 89,397 99,794 MPL®D 25 751,066 221,324 44,957 91,265 77,989 week 7 None 694,741 244,212 1,801 6,849 9,245 IL-12 5 177,438 17,232 20,2761 3,494 26,859 IL-12 1 183,571 44,213 21,246 5,063 13,447 IL-12 0.2 852,2921251,157 37,104 37,717 88,933 MPL®D 25 783, 6221 187, 0551 30,694 89,153 59,297 SUBSTITUTE SHEET rule 26 WO 99/40936 PTU9/24 PCTfUS99/02847 -22- Table 2: Opsonophagocytic activity of sera of mice immunized with PnPs-14-CRM 1 9 7 /AlpO 4 formulated with IL-12 or MPL® Bacteria_________Surviving_______ Week 5 Sera Week 7 Sera Initial Serum No 0.2 ug 1 ug 5 jg 25 pg No 0.2 ug 1l"g 5 ug 25 pg Dilution IL-12 IL-12 IL-12 IL-12 MPLO IL-12 IL-12 IL-12 IL-12 MPLO Tested 2 6 10 6 6 9 6 6 7 7 8 12 7 9 9 7 21 4 10 9 6 16 32 4 24 25 3 47 8 17 26 8 32 71 12 61 94 23 68 6 85 90 21 64 64 46 90 89 51 116 34 79 99 76 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -23- EXAMPLE 2: Nature of T helper cells induced by Pneumococcal conjugate vaccine (PnPs-14-

CRM

1 97 /A1PO 4 formulated with IL-12 STUDY DESIGN Groups of eight Balb/c mice were immunized subcutaneously at the base of the tail with 1 /g PnPs- 14-CRM, 97 conjugate formulated with 100 Ag A1PO 4 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 CRM 19 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-y, 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 the mice were bled once more.

Four days later their draining lymph node cells and splenocytes were harvested and cultured for six days with CRM 97 lysozyme, ConA or in medium alone. Culture supernatants from parallel cultures were harvested at day 3 and day 6 and assayed for IFN-y, IL-5 and IL-10 by

ELISA.

RESULTS

Formulating PnPs-14-CRM 97 /AlPO 4 vaccine with the lower doses of IL-12 (0.2 /g and 1.0 pg) greatly SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/S99/02847 -24enhanced the IgG2a and IgG3 responses to Pnl4 at week but not IgG1 (see Table 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 zg IL-12 dose cause the dramatic 10-fold) reduction in IgG1 titers relative to the control group without cytokine.

One week after immunization, lymph node cells from mice immunized without IL-12 produced IFN-y, IL-5 and when stimulated with CRM 197 in vitro (Table 4).

Adding IL-12 to the vaccine dramatically increased the antigen-specific production of IFN-y and abolished the ability of the lymphoid cells to produce IL-5 and Maximal IFN-y production was obtained with the lowest dose of IL-12 (0.2 pg); higher doses, particularly 5 ug, appeared to reduce the levels of this cytokine. This was most clearly seen in cultures stimulated with 1 yg/mL CRM 19 The reduction in IFN-y with higher doses of IL-12 may not reflect a generalized suppressive phenomenon since IFN-y 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-y in response to stimulation with CRM 9 compared to mice immunized without IL-12 (Table As observed 7 days after primary vaccination, 0.2 gg to 1.0 4g IL-12 were optimal doses of IL-12 for augmentation of an IFN-y response. In contrast, however, IL-5 and SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 production were differentially affected. The 1.0 and ug doses of IL-12 essentially eliminated the response but, by comparison, had only a minor effect on production. IL-12 (5.0 Mg) abolished the ability of splenocytes but not lymph node cells to produce (Tables 5 and 6).

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -26- Table 3: Effect of IL-12 on immune response to an alumbased PnPsl4 glycoconjugate vaccine in Balb/c Mice Week IL-12 Dose IgG IgG1 IgG2a IgG2b IgG3 3 none 41,480 7,347 1,387 895 2,333 0.2 26,253 1,521 1,118 171 5,911 1 26,124 966 2,155 248 5,991 10,753 541 671 183 3,242 none 234,220 33,284 2,896 3,105 2,487 0.2 674,996 71,808 18,245 6,789 107,470 1 632,714 32,022 22,749 7,853 44,350 224,832 19,495 10,083 1,287 25,212 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -27- Table 4: Cytokines produced by lymph node cells taken 7 days after single immunization with PnPs-14 conjugate formulated with A1PO 4 and IL-12 Day 6 Cultures Antigen No 0.2 ug 1.0 Mg 5.0 Mg Cytokine in vitro gg/ml IL-12 IL-12 IL-12 IL-12 IFN-y 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 Con A 1 43.8 97.1 107.1 105.4 Medium <0.75 3.6 10.6 5.2 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 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 SUBSTITUTE SHEET rule 26 WO 99/40936 PTU9/24 PCTIUS99/02847 -28- Table 5: Cytokine production by splenocytes two weeks after secondary vaccination with PnPs-14 conjugate f ormulated with A1P0 4 and IL-12 ___Day 6 Cultures Antigen No 0 .2 ug 1 .0 u~g 5. 0 A.sg Cytokine in vitro pug/ml IL-12 IL-12 IL-12 IL-12.

IFN-y CRM 30 7.0 98.4 83.2 50.9 (U/xnL) 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 CRM 30 13.2 3.1 0.6 <0.2 (ng/mL) CRMY 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 CRM 30 8.6 4 7.1 0.6 (ng/rnL) CRM 1 1.1 2.5 1.7 <0.2 Lysozyne 30 <0.2 <0.2 <0.3 <0.2 Con A 1 10.5 0.4 <0.3 <0.2 SUBSTITUTE SHEET rule 26) WO 99/40936 PTU9/24 PCT/US99/02847 -29- Table 6: Cytokine production by lymph node cells two weeks after secondary vaccination with PnPs-14 conjugate formulated with AlPO 4 and IL-12 ___Day 6 Cultures Antigen No 0.2 msg 1.0 /.zg 5.0 pg Cytokine in vitro pg/ml IL-12 IL-12 IL-12 IL-12 IFN-y CR14 30 9.8 86.9 58.7 62.0 (U/ML) CR14 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 CR14 30 12.5 1.4 <0.2 (ng/mL) CR14 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 CR14 30 11.3 9.9 7.2 3.6 (rig/mL) CR14 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 SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/US99/02847 EXAMPLE 3: IL-12 adjuvant activity with poorly immunogenic Pneumococcal conjugate Study Design Swiss-Webster mice (10 per group) were immunized with 1 pg Pnl8C conjugate formulated with or without 100 pg AlPO 4 The vaccines were supplemented with either IL- 12 1 or 5 pg), 100 pg MPL® or 20 pg QS-21

T

Normal mouse serum 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 CRM 1 9 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 pg of IL-12 caused a 3-fold rise in the IgG titer of pooled week 5 sera, whereas vaccine formulated with 1 pg of IL-12 appeared to induce no SUBSTITUTE SHEET rule 26 WO 99/40936 PCTfUS99/02847 -31- Pnl8C response. The lowest dose of IL-12 (0.1 pg) induced the same response as the AlPO 4 -formulated vaccine not containing IL-12. The vaccine formulated with MPL®/A1PO 4 induced the highest frequency of responses; 7/10 mice gave OD>0.2, in contrast to QS-21/AlPO 4 and AlPO 4 alone, each of which induced 4/10 responders. Mice immunized with vaccine containing IL-12 plus AlPO 4 induced 2/10, 0/10 and 1/10 responders at IL-12 doses of 0.1 ug, 1.0 ig, and 5 ug, respectively.

In this experiment MPL® and QS-21 T caused at most a 3- to 4-fold increase in the Pnl8C IgG response. In the absence of A1PO 4 IL-12 did not have a profound adjuvant effect on the Pnl8C IgG response. The vaccine containing a 1 pg 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 T appeared to enhance the Pnl8C IgG response.

Among the vaccines formulated without AlPO4, QS-21

T

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 CRM 1 9 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 CRM 197 IgG response in mice immunized with vaccine formulated without A1PO 4 Moreover, there was an IL-12 dose-dependent increase in both IgG1 and IgG2a titers at weeks 3 and 5, as well as SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -32an increase in IgG2b at week 5. The IgG1 and IgG2a titers at week 5 were similar to those induced by vaccine formulated with 100 pg 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 the increase in both IgG1 and IgG2a titers suggest that, within this model at least, IL-12 enhances priming of both TH-l-like and TH-2-like helper cells by PnOsl8C-CRM,, 9 conjugate in the absence of AlPO 4 When added to the Pnl8C conjugate vaccine formulated with A1PO 4 the 0.1 pg dose of IL-12 caused little if any increase in the week 3 total IgG response to CRM, 19 but a 3-fold increase at week 5. However, this dose of IL-12 increased the IgG2a titer at week promoting titers similar to that induced by vaccine containing MPL or QS-21. IL-12 did not markedly increase the IgG2b titers. As seen in previous experiments, higher doses of IL-12 resulted in a sharp decline in IgG titers with all subclasses being affected.

SUBSTITUTE SHEET rule 26) Table 7: Effect of IL-12 on IgG response to PnOsl8C conjugate IqG titer IcrG subclasses at week Ch2

I

Adjuvant (pg/dose) Wk3 Wk5 IgG1 IgG2a IgG2b IgG3 Al PO 2 <100 4,608 4,591 116 <100 <100 0.1 pg IL-12 AlPO 4 <100 3,681 1,472 265 259 450 pg IL-12 AlPO 4 <100 130 <100 <100 <100 <100 5.0 pg IL-12 AlPO 4 260 13,545 7,820 1,426 <100 1,481 100 pg MPL/A1P0 4 233 9,027 1,522 935 877 <100 QS-21 AlP0 4 <100 7,989 879 1,395 1,062 1,004 none 107 10,768 5,238 345 <100 144 0.1 pg IL-12 <100 1,808 336 105 <100 <100 pg IL-12 <100 22,257 12,443 671 172 773 pg IL-12 <100 460 203 <100 <100 400 100 pg MPL 112 1,729 524 363 189 126 QS-21 <100 3,573 2,483 101 <100 113 Table 8: Effect of IL-12 on CRM 97 IgG response three weeks after vaccination with PnOsl8C conjugate Adjuvant (pg/dose) IgG IgGi IgG2a AlPO 4 70,964 8,706 3,516 0.1 pg IL-12 AlPO 4 103,589 4,754 13,025 pg IL-12 AlPO 4 26,927 506 2,926 pg IL--12 AlPO 4 19,579 241 2,665 100 pg MPL®/AlPO, 651,315 92,245 79,508 QS-21 T M

/ALPO

4 572,255 116,583 38,419 None 7,630 452 1,023 0.1 jig IL-12 32,403 3,475 3,713 pg IL-12 60,987 4,615 5,951 pg IL-12 128,697 10,498 10,686 100 pg MPL®/TEM 462,289 40,010 24,979 QS-21

TM

556,440 111,533 53,799 Table 9: Effect of IL-12 on CRM197 IgG response five weeks after vaccination with PnOsl8C conjugate (two weeks after boosting) r I Adiuvant (ua/dose) laG I nOt TnO2~i

I

Ig IaG IaG' I I r,-

C,)

tTl r!1 Al PO 4 0.1 Pg IL-12 AlPO 4 Pg IL-12 AlPO, jig IL-12 AlPO 4 100 pg MPL®/AlPO 4 QS-2 1

TM

/ALPO

4 634,631 2,225, 000 105,765 71, 618 4,384,000 000, 000 102, 974 88,204 8,018 1, 582 637,655 000, 000 45,955 317, 083 12,598 13, 806 371, 652 873, 674 8,812 16, 869 1,096 744 111,646 144, 132 None 0. 1 pg 1. 0 pg 0 pg 100 pg QS-21 IL-12 IL-12 IL-12

MPL®/TEM

62,341 296, 791 1,026,060 1,367,771 4,173,765 12, 783 52,288 101,381 74,494 264,691 1,303,508 3, 655 23,741 96, 024 108, 815 266, 160 445,712 1, 679 7, 069 11,862 14, 258 303, 662 131,991 000, 000 WO 99/40936 PCT/US99/02847 -36- EXAMPLE 4: Effect of IL-12 on the IgG response of Swiss Webster mice to bivalent vaccine containing PnPs6B-CRM 19 7 and PnPs-14-CRM 197 STUDY DESIGN Swiss Webster mice were immunized subcutaneously at weeks 0 and 3 with a vaccine comprising 0.1 g per dose of PnPs6B-CRM 19 glycoconjugate (a conjugate of capsular polysaccharide from serotype 6B pneumococci covalently linked to CRM 197 plus 0.1 ig per dose of PnPsl4-CRM 19 glycoconjugate. The vaccines were administered with 0, 8, 40, or 200 ng IL-12, either alone or in combination with 100 ig alum (AlPO 4 Normal mouse serum was included as a carrier protein to stabilize the IL-12 at low concentrations. A control group of mice was immunized with the vaccine formulated with 100 g monophosphoryl lipid A The mice were bled at week 3 (primary response) and week 5 (secondary response). Sera were tested for IgG antibodies to Pn6B and Pnl4 capsular polysaccharide by ELISA.

RESULTS

Response to PnPs6B conjugate Table 10 illustrates the pooled serum IgG response to the Pn6B component of the bivalent vaccine. Little or no response to Pn6B was detected at week 3 if the vaccine contained no adjuvant or was formulated with only A1PO 4 The highest titers after a single vaccination appeared to SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -37be induced by vaccine containing either MPL® or 8-40 ng of IL-12 co-formulated with alum. These titers however were low, less than 3,000. The week 5 responses show that after boosting, vaccines formulated with 40 ng IL-12 plus A1PO 4 or with MPL® induced the highest IgG titers to Pn6B. In the absence of alum, IL-12 in the 8 to 200 ng dose range did not enhance the IgG titers to Pn6B.

The IgG subclass response to Pn6B at week 5 is shown in Table 10. The titers of the individual IgG subclasses were similar in mice immunized with vaccine containing no adjuvant or vaccine formulated with A1PO 4 (no IL-12).

Moreover, formulating the vaccines with 8-200 ng of IL-12 in the absence of AlPO 4 did not alter the IgG subclass response. In contrast, these doses of IL-12 when combined with AlPO 4 resulted in substantially increased IgG1 and IgG2a titers to Pn6B. These titers were similar to those obtained with vaccine formulated with MPL®. IL- 12 also increased the IgG2b and IgG3 titers induced by vaccine formulated with AlPO 4 however, these titers appeared to be substantially lower than those induced by vaccine formulated with MPL®.

To determine if the increases obtained with a combination of IL-12 and A1PO 4 were statistically significant, the Pn6B IgG titers of individual mice in selected groups were determined. The geometric mean titers (GMT) are presented in Table 11. The data indicate that groups immunized with vaccines formulated without adjuvant or with AlPO 4 alone had similar GMT against Pn6B. Formulating the vaccine with AlPO 4 plus SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/US99/02847 -38- IL-12 resulted in a 29-fold increase in titer over that induced by vaccine containing no adjuvant. When all the data were tested by ANOVA (analysis of variance by JMP software;-SAS Institute, Cary, North Carolina), no statistically significant differences were found. Upon comparison of subsets of data, ANOVA indicated a statistically significant difference when comparing the week 5 responses induced by vaccine containing no adjuvant and vaccines formulated with AlPO 4 and various doses of IL-12. Of these, the vaccine formulated with AlPO 4 plus 40 ng IL-12 induced a significantly higher Pn6B titer than vaccine formulated without adjuvant. As a further indication of the heightened immunogenicity of that formulation, 7 of the 10 mice in that group had Pn6B titers greater than or equal to 50,000 compared to only 1 and 2 mice each in the groups vaccinated with conjugate formulated without adjuvant or with AlPO 4 alone.

SUBSTITUTE SHEET rule 26) Table 10: Effect of IL-12 on the IgG response to PnPs6B in mice immunized with a bivalent PnPs6B/14 pneumococcal glycoconjugate vaccine Pn6B IgG Titer* Pn6B IgG Subclass Response at Week Group Adjuvant Week 3 Week 5 IgGi IgG2a IgG2b IgG3 P344 200 ng IL-12 630 161,867 19,474 22,664 2,954 7,333 P345 40 ng IL-12 2,609 429,006 61,364 24,172 4,117 9,830 P346 8 ng IL-12 AlPo 4 1,977 284,206 46,734 32,859 3,195 8,764 P347 AlP0 4 (no IL-12) 279 120,999 8,767 2,199 688 301 P348 200 ng IL-12 <100 22,401 6,816 3,147 501 1,104 P349 40 ng IL--12 164 23,343 5,056 2,532 879 292 P350 8 ng IL,-12 642 81,748 17,702 3,573 5,151 1,786 P351 None <100 20,153 3,061 1 1,506 364 1,220 P352 100 pig MPL® 2, 872 840,513 84,660 130,813 43,505 25,749 Pooled serum titers WO 99/40936 PTU9124 PCT/US99/02847 Table 11: Pn6B IgG titers of individual mice Group P344 P345 P346 P347 P350 P351 P352 A1PO 4 A1PO 4 AIPO, Mouse 200 ng 40Ong 8 ng lO8ng N10 g IL-2 I-1 IL12 (no IL-12) IL-12 Adjuvant mpL® 1 1,957 596,886 13,457 306,012 833,148 3,544 7,556 2 2,498 1,205 1,000,000, 3,653 9,431 326 1,359,470 3 100 9,453 1,422 8,708 3,163 1,1361 81 4 1 11,830 70,278 168,481 41,395 109,399 24,140 583,097 1,823 157,427 16,454 677,407 252 50,785 86,656 6 6,114 90,843 989 9,089 150,245 228 284 7 279 49,182 372,709 17,164 112 1,351 1,000,000 8 756,503 408,348 425 7,329 393 36,805 473,652 9 1,000,000 1,000,000 667,988 100 13,622 22,817 927,213 177 1,052,210 6,206 245 182,629 851 GMT 4,347 103,743 22,580 9,735 10,120 3,589 55,799 Mice with titer 2 7 4 2 4 1 61 000 Statistical Comparisons (ANOVA: a=0.05) AlPO 4 40 ng IL-12 vs. no adjuvant: significant SUBSTITUTE SHEET ruie 26 WO 99/40936 PCT/US99/02847 -41- Response to PnPsl4 conjugate The IgG response to the PnPsl4 component of the vaccine is shown in Table 12. The data indicate that IL-12 in the 8-40 ng dose range, either alone or when formulated with A1P04, did not enhance the response to PnPsl4 after primary or secondary vaccination.

Moreover, subclass analysis indicated that IL-12 did not enhance the IgG2a titers when formulated with IL-12. In this study, MPL® did not have the profound adjuvant effect on the PnPsl4 response that was observed in previous studies, at least when assaying pooled sera.

To get an idea of the degree of variation of the response of each group, individual sera were assayed for Pnl4 IgG antibodies at a 1/300 dilution. The results presented in Table 13 suggest that there was a large range of responses in each group, the Coefficient of Variation (CV) ranged from 0.229 to 0.587, with the exception of the group immunized with vaccine containing MPL® where the CV was 0.051. Thus, it appeared that MPL®, but not IL-12, may have acted as an adjuvant for the Pnl4 IgG response and reduced the mouse-to-mouse variation.

SUBSTITUTE SHEET rule 26 Table 12: Effect of IL-12 on the IgG subclass response to Pnl4 in mice immunized with a bivalent PnPs6B/14 pneumococcal glycoconjugate vaccine PnPsl4 Ig* Titer* PnPsl4 IgG Subclass at Week Adjuvant Week 3 Week 5 IgG1 IgG2a IgG2b IgG3 200 ng IL-12 2 2 2,170 58,657 6,880 8,996 1,945 5,995 A1PO 4 ng IL-12 AlPO 1,641 53,557 8,646 3,003 3,684 2,745 A1PO 4 8 ng IL-12 8 2 2,181 85,173 10,094 11,346 5,328 2,560 A1PO 4 A1PO 4 (no IL-12) 2,102 201,082 54,989 4,030 6,402 3,745 200 ng IL-12 849 18,293 5,769 1,582 536 799 ng IL-12 1,544 11,442 4,350 714 514 455 8 ng IL-12 113 12,169 5,286 354 245 330 None 509 22,601 6,080 808 618 694 100 pg MPL® 18,616 77,106 15,745 4,275 10,205 3,916 *Pooled serum titers "0 0 \o Table 13: Response of individual mice to Pnl4 component of Pn6B/Pnl4 bivalent pneumococcal conjugate vaccine* Standard Coefficient of Adjuvant O.D. Range O.D. Mean Deviation Variation A1PO, 200 ng IL-12 0.034-0.990 0.788 0.318 0.404 AlP0 4 40 ng IL-12 0.457-0.948 0.771 0.176 0.229 AlPO 4 8 ng IL-12 0.023-0.923 0.707 0.281 0.397 A1PO,, (no IL-12) 0.328-0.974 0.770 0.220 0.285 8 ng IL-12 (no alum) 0.009-0.812 0.505 0.292 0.587 No adjuvant 0.030-0.876 0.614 0.343 0.558 100 jig MPL®D 0.791-0.918 10.863 10.044 1 0.051 *Individual sera tested by ELISA at 1/300 dilution for IgG antibodies to Pnl4.

WO 99/40936 PCT/US99/02847 -44- EXAMPLE 5: Comparison of the effect of IL-12 in the presence or absence of alum on the murine immune response to monovalent PnPsl4- CRM1 97 conjugate vaccine STUDY DESIGN BALB/c mice (8 per group) were immunized subcutaneously at week 0 with 1 ig PnPsl4-CRM 19 conjugate formulated with or without 100 pg AlPO 4 and either no IL-12 or with 8, 40, 200, 1,000 or 5,000 ng IL-12. Normal mouse serum was included as a carrier protein to stabilize IL-12 at low concentrations. At week 1, lymph node cell suspensions were prepared from half the mice in each group and evaluated for antigen-specific cytokine production in vitro. Their spleens were also harvested and weighed.

At week 3 the remaining mice were bled and re-immunized with the same vaccine formulation used in the initial vaccination. At week 5 the twice-immunized mice were bled, their spleens weighed and their splenocytes evaluated for cytokine production. PnPsl4 and CRM 197 IgG and IgG subclass titers were determined on pooled sera.

When the assays were performed using sera from individual mice, the results are expressed as geometric mean titers (GMT).

RESULTS

Effect of IL-12 on spleen weight one week after immunization SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 One week after the first immunization, mice receiving 5,000 ng IL-12, but not lower doses of IL-12, in the absence of AlPO 4 had significantly higher spleen weights than those receiving vaccine containing neither alum nor IL-12 (Table 14). Vaccines containing AlPO 4 induced higher spleen weights when formulated with 40 to 5000 ng IL-12. Pair-wise comparisons indicated that vaccines formulated with 200 or 1000 ng IL-12 plus AlPO 4 induced higher spleen weights than those formulated with the same dose of IL-12 in the absence of AlPO 4 Overall, the data indicate that formulating IL-12 with AlPO 4 greatly enhanced a biological activity of the cytokine, its ability to cause increased spleen weight one week after vaccination.

Effect of IL-12 on the IgG response to PnPsl4 Initially, pooled sera were assayed for IgG antibodies to PnPsl4 (Table 15). The clearest indication of an adjuvant effect was noted after primary immunization with vaccine containing AlPO 4 and 8 to 40 ng IL-12. This combination resulted in a 17- to 21-fold increase in the IgG titer relative to mice immunized with vaccine formulated with neither AlPO 4 nor IL-12.

The combination of AlPO 4 and IL-12 resulted in higher responses than when used individually; on their own A1PO 4 and the 40 ng dose of IL-12 caused 4-fold and increase in week 3 IgG titers, respectively. Analysis of individual sera from mice immunized with A1PO 4 containing vaccines (Table 16) showed that 8 ng IL-12 induced 5-fold higher PnPsl4 IgG titer after primary vaccination than vaccine adjuvanted with only AlPO 4 The SUBSTITUTE SHEET rule 26 WO 99/40936 PCT[US99/02847 -46difference in titers was statistically significant.

Higher doses of IL-12 did not enhance the response. The 1,000 to 5,000 ng doses of IL-12 caused a marked decline in PnPsl4 IgG titers. After the second immunization only the 40 ng dose of IL-12 caused a significant rise (3-fold) in the PnPsl4 titer induced by the AlPO 4 -based vaccine.

The pooled serum data suggest that the combination of A1PO 4 and 8-40 ng IL-12 enhanced the IgGl titers after primary immunization. After two vaccinations, IL-12 did not enhance the IgG1 titers to PnPsl4 in mice immunized with conjugate in the absence of AlPO 4 as indicated by analysis of pooled (Table 15) and individual sera (Table 17). Moreover, among mice immunized with vaccine containing AlPO 4 the addition of 8 to 200 ng IL-12 did not result in higher IgG1 titers after 2 vaccinations (Table 17).

The most profound effect of IL-12 was to substantially increase the PnPsl4 IgG2a response at week 5. This was seen both when the vaccine contained A1PO 4 or was formulated without A1P0 4 (Table 18). In the absence of AlPO 4 statistically significant increases (14- to 42-fold) in IgG2a GMT were obtained with 8 to 1,000 ng IL-12. Similarly, 8-1,000 ng IL-12 enhanced the ability of AlPO 4 -containing vaccines to induce IgG2a antibodies, although in this study only the titers induced by the 8 and 40 ng doses of IL-12 were statistically higher. Overall, the highest IgG2a titers were induced by conjugate formulated with A1PO 4 and 40 ng IL-12. This was significantly different from the IgG2a SUBSTITUTE SHEET rule 26 WO 99/40936 PCTIUS99/02847 -47titers induced by 40 ng IL-12 in the absence of AlPO 4 again indicating that the adjuvant activity of IL-12 was enhanced by alum.

IgG2b and IgG3 titers were assayed on pooled sera only (Table 15). Doses of IL-12 in the range of 8 to 1,000 ng when co-formulated with AlPO 4 but not in its absence, promoted substantial increases in IgG3 titers after primary and secondary immunization. No consistent effect of IL-12 on the IgG2b titers was noted.

Effect of IL-12 on the IgG response to CRM 19 The IgG response to CRM 19 was also evaluated to see if there were differences 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 40 ng IL-12 appeared to modestly increase the IgG titers to

CRM

97 after two vaccinations. However, the highest IgG titers to CRM 197 were obtained when the vaccine was formulated with both AlPO 4 and 8-40 ng IL-12. The heightened adjuvant activity of IL-12 co-formulated with A1PO 4 is indicated by the finding that, on their own, ng IL-12 and AlPO 4 resulted in 6-fold and 17-fold increases in IgG titer at week 5, but when combined together the increase was 147-fold. IL-12 enhanced the IgG1 response to CRM, 97 regardless of whether the vaccine was formulated with or without A1PO 4 (Tables 19 and IL-12 substantially increased the week 5 IgG2a titers to

CRM

19 after immunization with vaccines containing AlPO 4 (Table 19). Again the optimal dose of IL-12 appeared to SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -48be 40 ng. The cytokine appeared to increase the IgG2b titers induced by vaccine containing A1PO 4 Effect of IL-12 on cytokine profile of CRM 197 -specific T cells Cytokine production by spleen cells taken two weeks after secondary vaccination (week 5) revealed effects of IL-12 on the priming of both IFN-y and IL-5 producing cells. Splenocytes from mice immunized in the absence of A1PO 4 and IL-12 produced detectable levels of but not IFN-y, when stimulated with CRM1,, in vitro (Table 21). Formulating the vaccine with IL-12 appeared to enhance the induction of IL-5-producing cells with peak activity occurring with 40 ng of the cytokine.

Higher doses of IL-12 resulted in decreased production of IL-5, with virtually no cytokine being produced by mice immunized with conjugate vaccine containing 1,000 to 5,000 ng IL-12. Convincing IFN-y production was detected only from the splenocytes of mice immunized with vaccines formulated with 5,000 ng IL-12. When the vaccine was formulated with A1PO 4 the addition of 8 ng IL-12 resulted in priming of cells that produced copious amounts of IFN-y, whereas in the absence of the cytokine only antigen-specific IL-5 production was detected. It appears that priming for maximal IFN-y production occurs with 40 to 1,000 ng IL-12. Addition of 5,000 ng IL-12 abolished the ability of the vaccine to prime for producing cells.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -49- Table 14: Spleen weights of Balb/c mice one week after subcutaneous immunization with 1 ig PnPsl4-CRM 197 conjugate formulated with or without 100 ug A1PO 4 and the indicated doses of IL-12 Adjuvant Formulation Spleen Weight (grams) Standard Group Code IL-12 (ng) AlPO 4

AVERAGE

Deviation P641 0 0.179 0.0225 P642 8 0.148 0.0112 P643 40 0.162 0.0202 P644 200 0.175 0.0431 P645 1,000 0.196 0.0068 P646 5,000 0.357 0.0247 P647 0 0.151 0.0158 P648 8 0.151 0.0332 P649 40 0.217 0.0596 P650 200 0.290 0.0226 P651 1,000 0.277 0.0919 P652 5,000 0.305 0.0545 Statistical Comparisons (ANOVA; a=0.05) P642, P643, P644, P645 vs P641: not significant P646 vs P641: significant P648 vs P647: not significant P649, P650, P651, P652 vs P647: significant P641 vs P647: not significant P644 vs P650: significant P642 vs P648: not significant P645 vs P651: significant P643 vs P649: not significant P646 vs P652: not significant SUBSTITUTE SHEET rule 26 Table 15: Anti-PnPsl4 IgG response in Balb/c mice immunized with PnPsl4-CRM 197 conjugate formulated with IL-12 and AlPO 4

U)

Cni to P-i

ON

Id 0Fj PnPsl4 IgG Titers of Pooled Sera Adjuvant IgG IgG1 IgG2a IgG2b IgG3 Formulation IL-12 (ng) A1PO 4 Week 3 Week 5 Week 3 Week 5 Week 3 Week 5 Week 3 Week 5 Week 3 Week 0 1,691 24,498 479 9,967 139 492 <100 <100 295 1,516 8 4,679 32,966 841 9,860 377 1,902 108 609 390 1,354 6,484 50,096 1,235 17,631 207 1,209 58 724 1,633 4,017 200 5,330 51,240 385 7,568 715 3,748 290 1,397 1,091 4,51S 1,000 6,347 69,673 1,286 12,814 859 6,532 124 <100 782 6,20E 5,000 1,131 19,621 229 3,598 126 1,392 <100 <100 635 3,616 0 7,825 103,092 1,714 38,147 195 1,535 617 3,973 447 2,963 8 29,506 195,069 7,444 58,046 1,207 6,697 693 4,843 5,669 25,407 35,567 295,361 4,945 46,030 2,883 17,267 1,371 9,911 5,797 22,602 200 10,177 190,701 1,777 41,800 626 9,816 <100 1,479 3,443 23,64E 1,000 2,422 245,683 90 31,373 167 13,847 <100 722 1,173 34,039 5,000 1,304 35,333 91 5,228 <100 1,429 <<10 <100 772 8,065 0 Table 16: Effect of IL-12 on the IgG response to PnPsl4 in mice immunized with PnPsl4-CRM, 7 conjugate formulated with AlPO, PnPsl4 IgG GMT (fold increase) Group IL-12 (ng) AlPO 4 Week 3 Week P647 0 3,037 27,027 P648 8 16,68 55,855 (2.1) P649 40 6,667 88,271 (3.4) P650 200 2,333 57,076 (2.1) P651 1,000 611 30,886 (1.1) P652 5,000 617 10,989 (0.4) Statistical Comparisons (ANOVA; -=0.05) Week 3 titers P648 vs P647: significant P651 vs P647: significant P649, P650, P652 vs P647: not significant Week 5 titers P649 vs P647: significant P648, P650, P651 vs P647: not significant 00

-J

WO 99/40936 PCT/US99/02847 -52- Table 17: PnPsl4 IgG1 titers in mice twice immunized with PnPsl4-CRM 1 97 conjugate vaccine formulated with or without A1PO 4 and various doses of IL-12 Adjuvant Formulation IgG1 GMT Group Code IL-12(ng) AlPO 4 (Geometric Mean Titer) P641 0 9,492 P642 8 5,964 P643 40 14,028 P644 200 4,628 P645 1,000 5,815 P646 5,000 1,757 P647 0 15,283 P648 8 35,730 P649 40 31,855 P650 200 34,166 P651 1,000 15,347 P652 5,000 4,022 Statistical Comparisons (ANOVA; a=0.05) P642, P643, P644, P645, P646, P647, P651 vs P641: not significant P648, P649, P650 vs P641: significant P648, P649, P650, P651 vs P647: not significant P652 vs P647: significant SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -53- Table 18: PnPsl4 IgG2a titers in mice twice immunized with PnPsl4-CRM, 9 conjugate vaccine formulated with or without A1PO 4 and various doses of IL-12 Group Code IL-12 (ng) AlPO 4 IgG2a GMT at Week (Fold Increase*) P641 0 97 P642 8 1,418 (14.6) P643 40 1,509 (15.6) P644 200 2,228 (23.0) P645 1,000 4,126 (42.5) P646 5,000 289 P647 0 806 P648 8 6,841 P649 40 13,252 (16.4) P650 200 4,740 (5.9) P651 1,000 3,291 (4.1) P652 5,000 368 relative to control vaccines not containing IL-12 Statistical Comparisons (ANOVA; a=0.05) P642, P643, P644, P645 vs P641: significant P646 vs P641: not significant P648, P649 vs P647: significant P650, P651, P652 vs P647: not significant P643 vs P649: significant SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -54- Table 19: Anti-CRM,, IgG response in immunized with PnPsl4-CRM 97 conjugate 12 and AlPO 4 Balb/c mice formulated with IL- Adjuvant CRM 9 IgG Titer IgG Subclasses at (Pooled Sera) Week 5 (Pooled Sera) IL-12 (ng) A1PO 4 Week 3 Week 5 IgG1 IgG2a IgG2b 0 3,843 8,965 703 1,269 792 8 2,456 14,389 4,674 <100 <100 3,200 53,758 14,073 3,403 <100 200 1,666 13,419 1,803 2,044 <100 1,000 4,999 3,663 <100 506 <100 5,000 2,841 3,641 <100 <100 <100 0 4,870 153,075 55,922 1,796 407 8 89,558 1,515,87 377,82 85,972 10,972 19,566 1,319,10 147,03 199,29 7,206 200 6,884 315,071 48,852 36,807 3,865 1,000 7,292 545,827 126,72 44,190 4,127 5,000 7,213 7,029 1,041 769 <100 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 Table 20: IgG1 titers to CRM, 19 in Balb/c mice immunized with PnPsl4-CRM 19 7 conjugate formulated with IL-12 and A1PO 4 Group Code IL-12 (ng) AlPO 4 IgG1 GMT Fold Increase P641 0 317 P642 8 1,136 3.6 P643 40 9,141 28.8 P644 200 1,627 5.1 P645 1,000 100 0.3 P646 5,000 174 0.6 P647 0 22,061 P648 8 119,130 5.4 P649 40 73,226 3.3 P650 200 14,391 0.7 P651 1,000 33,468 P652 5,000 317 0.01 Statistical Comparisons (ANOVA; a=0.05) P643, P644 vs P641: significant P642, P645, P646 vs P641: not significant P648, P649, P650, P651 vs P647: not significant P642 vs P648, P643 vs P649, P644 vs P650, P645 vs P651: significant SUBSTITUTE SHEET rule 26 Table 21: Cytokine production by splenocytes from mice immunized twice with PnPsl4- CRM1 97 formulated with IL-12 in the presence and absence of AlPO 4 04 En Cells Stimu- IL-12 Dose in Vaccine Formulated IL-12 Dose in Vaccine Formulated lated With Without APO 4 (ng) With AlPO, Cytokine Antigen ug/mL 0 8 40 200 1,000 5,000 0 8 40 200 1,000 5,000 IFN-y CRM,97 30 <0.06 <0.06 0.4 0.3 0.4 6.9 0.1 20.6 32.1 30.2 29.4 21.0 (U/mL) CRM1i, 10 <0.06 <0.06 0.3 0.2 0.3 3.4 0.1 16.5 31.7 30.2 27.6 21.0 CRM1 7 3 <0.06 <0.06 <0.06 <0.06 0.2 1.4 <0.03 15.7 30.9 28.7 26.7 20.1 CRMi 1 <0.06 <0.06 <0.06 <0.06 0.2 0.4 <0.03 12.8 30.9 28.2 27.9 18.0 CRM,9 0.3 <0.06 <0.06 <0.06 <0.06 0.1 0.1 <0.03 6.3 27.5 26.2 26.5 Lysozyme 30 <0.06 <0.06 <0.06 <0.06 <0.03 <0.03 <0.03 <0.0 <0.0 <0.0 <0.02 <0.02 Con A 1 11.9 15.4 15.8 15.9 20.6 21.0 21.1 17.0 20.8 13.7 23.4 17.7 Medium <0.06 <0.06 <0.06 <0.06 <0.03 <0.03 <0.03 <0.0 <0.0 <0.0 <0.02 <0.02 CRM^., 30 370 480 2560 960 60 70 2010 1440 5280 1640 880 (pg/mL) CRM,,, 10 150 300 1110 260 <24 <24 1220 920 4590 410 430 CRM 7 3 30 90 910 200 <24 <24 1190 690 2830 1030 200 CRM, 1 <4 50 200 40 <24 <24 880 400 2150 520 140 CRMg, 7 0.3 <4 30 70 <4 <24 <24 670 180 440 270 90 Lysozyme 30 <4 <4 <4 <4 <24 <24 <24 <24 <10 <10 <10 Con A 1 <4 <4 <4 <4 <24 <24 60 80 40 <10 <10 Medium <4 <4 <4 <4 <24 <24 <24 <24 <10 <10 <10 WO 99/40936 PCTIUS99/02847 -57- EXAMPLE 6: Effect of IL-12/AlPO 4 on the humoral response to a nonavalent pneumococcal glycoconjugate vaccine STUDY DESIGN Evaluation of the effect of IL-12 on the IgG response to pneumococcal glycoconjugate vaccine was extended to a nonavalent vaccine composed of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F.

Swiss Webster mice were immunized with 0.1, 1, or jg of vaccine (carbohydrate weight) at weeks 0 and 3. The vaccine was administered alone, with AlPO 4 (100 4g) or with A1PO 4 admixed with 50, 200 or 1,000 ng of IL-12. Normal mouse serum was not included in the vaccine. The IgG responses to serotypes 4, 6B, 9V, 14, 18C and to the carrier protein CRM 19 were evaluated at week 5 2 weeks after boosting) by ELISA.

RESULTS

Response to CRM 19 at week Addition of IL-12 to a vaccine containing A1P04 resulted in a dose-dependent increase in IgG2a and IgG2b antibodies to CRM,,. This was seen at all doses of conjugate tested (Table 22). Increased IgG2a titers were evident in mice receiving 50 ng of the cytokine and were maximal at 1,000 ng. This SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -58contrasts with other studies where maximal IgG2a titers were obtained with 40-100 ng of cytokine added to the alum-based vaccine and where higher doses of IL-12 resulted in a diminished immune response. The reason for the differences in dose response between studies is not known. It may relate to differences in the vaccine, i.e., multivalent versus monovalent or that normal mouse serum included in the vaccine in previous studies to stabilize the cytokine at low concentrations was omitted.

Response to pneumococcal polysaccharides Formulating the nonavalent vaccine with A1P04 enhanced the IgG response to several serotypes including PnPs4, PnPs6B, PnPs9V and PnPsl4, especially when the lowest dose of conjugate (0.1 ug) was used (Tables 24-27). Addition of IL-12 did not appear to further enhance the IgG response to these serotypes. In the case of the PnPsl8C response, however, addition of 50 or 1,000 ng IL-12 to 5 ug of vaccine containing A1P04 resulted in higher geometric mean IgG titers to this serotype and higher proportion of mice with PnPs18C IgG titers above 10,000 (Table 23). The responses to PnPsl, 5, 19F and 23F were not evaluated.

Addition of IL-12 to nonavalent vaccines containing A1P04 resulted in dose-dependent increases in IgG2a titers to PnPs4, PnPs6B, PnPs9V SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -59and PnPsl4 (Tables 24-27). Generally, the increase in IgG2a paralleled that for the CRM, 97 response with highest titers being obtained with 1,000 ng of IL- 12. In contrast to the experiments using monovalent PnPsl4 conjugate or bivalent PnPs6B/PnPsl4 vaccine, the 50 ng dose of IL-12 had little or no effect on the IgG2a response to these serotypes. The exception is the IgG2a response to PnPsl4, as this dose of cytokine appeared to enhance the response to this serotype (Table 27).

Overall, this study indicates that IL-12 will promote the complement-fixing IgG2a antibody subclass response to multiple pneumococcal serotypes present in a multivalent vaccine.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 Table 22: Effect of IL-12 on the CRM 19 response of mice immunized with nonavalent pneumococcal glycoconjugate vaccine formulated with AlP0 4 Vaccine Formulation CRM, 9 7 Response at Week Conjugate IL-12 A1P04 IgG IgGI IgG2a IgG2b IgG3 (ng) (11g) Dose (ug) none none 391,021 138,784 1,687 3,277 102 0 100 1,419,910 609,704 4,328 11,349 181 50 100 3,226,410 896,621 27,736 18,086 728 5.0 200 100 2,991,990 584,991 87,732 28,855 2,937 1,000 100 16,224,900 906,192 303,656 87,726 3,023 none none 545,046 162,757 1,178 9,213 358 0 100 956,584 338,751 1,284 4,118 306 50 100 1,936,170 370,961 6,677 31,982 931 1.0 200 100 4,788,500 660,082 187,034 36,785 1,065 1,000 100 12,404,500 644,151 533,065 69,185 1,176 0.1 none none 15,215 3,800 <100 <100 <100 0.1 0 100 561,952 157,362 1,437 7,744 <100 0.1 50 100 807,363 141,670 16,064 26,978 2,092 0.1 200 100 1,560,380 313,263 38,686 51,737 306 0.1 1,000 100 2,296,310 202,111 112,158 36,958 1,054 Mice were immunized with the indicated dose of nonavalent pneumococcal glycoconjugate vaccine at weeks 0 and 3.

The conjugates were formulated alone, with A1P0 4 (100 ug) or with AlP0 4 plus IL-12. Sera from the week 5 bleed were analyzed for IgG antibodies to CRM1 9 SUBSTITUTE SHEET rule 26) WO 99/40936 PCTIS99/02847 -61- Table 23: Effect of IL-12 on the response to PnPsl8C in mice immunized with 5 ug nonavalent pneumococcal glycoconjugate vaccine formulated with A1P04 Vaccine Formulation PnPsl8C Response Conjugate IL-12 AlPO 4 IgG Titer Mice With Titer Dose (ug) (ng) (ug) (GMT) >10,000(n=5) 0 0 2,933 1 50 100 23,725 4 1,000 100 48,375 Mice were immunized with the indicated dose of nonavalent pneumococcal glycoconjugate vaccine at weeks 0 and 3.

The conjugates were formulated alone, with A1P0 4 (100 Pg) or with AlPO 4 plus IL-12. Individual mouse sera from the week 5 bleed were analyzed for IgG antibodies to PnPsl8C.

SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/US99/02847 -62- Table 24: Effect of IL-12 on the PnPs4 response of mice immunized with nonavalent pneumococcal glycoconjugate vaccine formulated with A1P04 Vaccine Formulation PnPs4 Response at Week Conjugate IL-12 A1PO 4 IgG IgGl IgG2a IgG2b IgG3 Dose (jug) (ng) (11g) none none 55,068 13,731 <500 <500 <500 0 100 233,008 55,620 <500 1,157 990 50 100 285,806 64,493 1,050 1,329 2,634 200 100 203,236 56,654 1,789 692 2,693 1,000 100 371,329 35,778 4,048 1,080 3,820 none none 77,714 9,070 <500 608 <500 0 100 141,371 14,829 <500 <500 542 50 100 97,999 14,336 449 814 1,034 200 100 137,674 17,380 752 569 816 1,000 100 214,739 25,056 4,685 1,260 4,055 0.1 none none 4,726 706 <500 <500 <500 0.1 0 100 79,686 12,071 <500 869 <500 0.1 50 100 70,917 9,649 1,032 1,389 <500 0.1 200 100 46,503 7,799 885 1,056 572 0.1 1,000 100 87,762 6,788 1,725 <500 1,682 Mice were immunized with the indicated dose of nonavalent pneumococcal glycoconjugate vaccine at weeks 0 and 3.

The conjugates were formulated alone, with AlP04 (100 pg) or with AlP04 plus IL-12. Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs4.

SUBSTITUTE SHEET rule 26) WO 99/40936 PCT/US99/02847 -63- Table 25: Effect of IL-12 on the PnPs6B response of mice immunized with nonavalent pneumococcal glycoconjugate vaccine formulated with A1P04 Vaccine Formulation PnPs6B Response at Week Conjugate IL-12 A1PO 4 Total IgG1 IgG2a IgG2b IgG3 Dose (ng) (0g) IgG none none 64,734 20,221 <100 195 325 0 100 103,686 39,061 138 2,498 1,801 50 100 487,798 127,753 916 3,200 13,758 200 100 214,743 59,979 924 959 6,459 1,000 100 427,514 94,478 4,426 2,552 13,142 none none 165,588 37,646 <100 2,047 2,337 0 100 730,920 133,441 990 2,770 7,468 50 100 428,549 77,124 838 3,755 12,931 200 100 164,820 29,685 316 662 4,703 1,000 100 401,513 51,132 11,44 2,735 31,613 2 0.1 none none 4,787 1,034 <100 <100 <100 0.1 0 100 370,177 71,287 603 11,372 5,712 0.1 50 100 137,091 25,447 1,029 3,346 3,411 0.1 200 100 128,428 31,634 434 2,698 1,891 0.1 1,000 100 524,385 67,301 9,611 11,587 8,711 Mice were immunized with the indicated pneumococcal glycoconjugate vaccine at dose of nonavalent weeks 0 and 3.

The conjugates were formulated alone, with AlP04 (100 pg) or with A1P04 plus IL-12. Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs6B.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT[S99/02847 -64- Table 26: Effect of IL-12 on the PnPs9V response of mice immunized with nonavalent pneumococcal glyconconjugate vaccine formulated with A1P04 Vaccine Formulation PnPs9V Response at Week Conjugate IL-12 A1PO 4 Total IgG1 IgG2a IgG2b IgG3 Dose (fg) (ng) (mg) IgG none none 36,831 15,568 306 250 317 0 100 78,614 37,544 359 667 286 50 100 117,345 61,031 1,073 834 2,089 200 100 134,333 35,031 2,973 748 2,594 1,000 100 197,407 40,368 15,353 2,147 1,945 none none 81,932 34,845 546 2,232 735 0 100 100,448 55,608 660 1,274 699 50 100 157,316 47,285 1,084 2,036 4,730 200 100 154,672 48,318 1,765 860 2,044 1,000 100 168,614 54,223 10,037 1,469 3,006 0.1 none none <500 181 <100 <100 <100 0.1 0 100 86,952 26,425 206 485 1,285 0.1 50 100 20,746 6,381 579 726 353 0.1 200 100 19,966 5,501 778 325 235 0.1 1,000 100 50,219 3,511 1,290 1,036 714 Mice were immunized with the indicated dose of nonavalent pneumococcal glycoconjugate vaccine at weeks 0 and 3.

The conjugates were formulated alone, with A1P04 (100 ug) or with a A1P04 plus IL-12. Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs9V.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 Table 27: Effect of IL-12 on the PnPsl4 response of mice immunized with nonavalent pneumococcal glyconconjugate vaccine formulated with A1P04 Vaccine Formulation PnPsl4 Response at Week Conjugate IL-12 AlPO 4 Total IgG1 IgG2a IgG2b IgG3 Dose (pg) (ng) (Gg) IgG none none 2,676 1,750 <100 <100 <100 0 100 11,792 15,704 124 580 1,723 50 100 56,712 31,056 6,144 2,854 11,840 200 100 5,049 3,050 1,588 <100 2,106 1,000 100 11,848 3,760 1,853 366 2,035 none none 4,846 3,116 <100 409 699 0 100 20,605 31,022 291 2,383 9,286 50 100 8,338 4,722 1,354 715 10,079 200 100 5,618 3,252 1,014 <100 583 1,000 100 13,026 3,551 2,879 671 2,070 0.1 none none <100 105 <100 <100 <100 0.1 0 100 114 392 <100 <100 710 0.1 50 100 2,140 2,838 <100 245 3,592 0.1 200 100 2,200 426 <100 622 759 0.1 1,000 100 394 378 219 100 658 Mice were immunized with the indicated dose of nonavalent pneumococcal glycoconjugate vaccine at weeks 0 and 3.

The conjugates were formulated alone, with A1P04 (100 pg) or with AlP04 plus IL-12. Sera from the week 5 bleed were analyzed for IgG antibodies to PnPsl4.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -66- EXAMPLE 7: The effect of IL-12 and A1P04 on the immune response to Neiserria meningitidis type C (menC) glyconconjugate vaccine STUDY DESIGN This study evaluated IL-12 with a vaccine against Neiserria meningitidis type C (menC). Swiss Webster mice were immunized at weeks 0 and 3 with 0.1 4g or 1 pg of MenC glycoconjugate formulated alone, with A1PO 4 (100 Pg) or a combination of IL-12 (50 ng) and A1P04. Normal mouse serum was not added to the vaccine. Mice were bled at weeks 3 and 5 and sera analyzed for IgG antibodies to menC polysaccharide by ELISA.

RESULTS

When immunized with the higher dose of conjugate, equivalent menC IgG titers were generated regardless of the adjuvant formulation. The addition of IL-12/A1P04 to the vaccine, however, resulted in higher IgG2a titers to the polysaccharide than if formulated with A1P04 (but no IL-12) or no adjuvant.

In mice immunized with the lower dose of conjugate, higher mening C titers were obtained when the vaccine was formulated with AlP04 (Table 28). The addition of IL-12 to the adjuvant did not enhance the overall IgG titer but did result in a >10-fold increase in IgG2a antibodies.

These data show that IL-12 in combination with A1P04 can promote the induction of complement-fixing IgG subclasses to menC glyconconjugate vaccine.

SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -67- Table 28: Effect of IL-12/A1PO 4 on the IgG response to menC glyconconjugate vaccine MenC Response Vaccine Formulation IgG IgG Subclass at Week MenC IL-12 AlPO 4 Week 3 Week 5 IgG1 IgG2a IgG2b IgG3 Conjugate (ng) (Og) (Ag) 50 100 33,176 598,027 83,662 7,218 4,351 1,436 0 100 34,553 404,111 71,017 1,383 3,085 1,006 0 0 16,254 288,493 63,043 1,965 <100 502 0.1 50 100 2,584 68,678 9,604 3,440 1,967 512 0 100 8,174 30,450 6,532 288 429 <100 0 0 1,724 7,894 1,767 <100 <100 <100 SUBSTITUTE SHEET rule 26) WO 99/40936 PCT[US99/02847 -68- Example 8: The effect of IL-12 and A1P04 on the immune response to Hemophilus influenzae type b glyconconjugate vaccine (HbOC) STUDY DESIGN This study evaluated IL-12 with a vaccine against Hemophilus influenzae type b. Swiss Webster mice (10 per group) were immunized at weeks 0 and 3 with 0.1 pg or pg of glyconconjugate vaccine consisting of capsular polysaccharide from Hemophilus influenzae type b (HibPs) conjugated to CRM 19 The vaccine (HbOC) was administered alone or in combination with A1P04 (100 pg) or a mixture of IL-12 (50 ng) plus A1P04. Normal mouse serum was not added to the vaccine. The mice were bled at weeks 3 and The antibody response to HibPs was measured using a Farr assay which measures all antibodies binding to the saccharide regardless of isotype, IgM, IgG and IgA.

The IgG subclass response was measured by ELISA.

Additionally, the IgG and IgG subclass response to CRM., 1 was also determined by ELISA.

RESULTS

The titers of anti-HibPs antibodies in serum pooled from the week 3 bleed (primary response) were not different between mice immunized with vaccine formulated alone, with A1P04 or IL-12 plus A1P04 regardless of the dose of conjugate used for immunization (Table 29).

Analysis of pooled serum from the week 5 bleed suggested SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -69that in mice immunized with 1 pg of HbOC with IL-12 plus alum resulted in at least a 10-fold higher anti-HibPs than when given with alum or without adjuvant (Table However, analysis of individual mouse sera showed that this was due to a single mouse having a titer of approximately 10,000 pg/mL. When the results are expressed as geometric mean titer there was no evidence of an enhanced HibPs response due to IL-12. The IgG subclass response to HibPs was evaluated on pooled sera by ELISA. The combination of IL-12 and A1P04 appeared to enhance the IgG2a titer 3-fold in mice immunized with 1 pg of conjugate. However, this was no different than the titer obtained with vaccine adjuvanted with A1P04 alone.

In mice immunized with 0.1 pg of HbOC, IL-12 plus A1P04 did not enhance the IgG2a titer to HibPs. That the IL- 12/A1P04 adjuvant combination was active was revealed by analysis of the anti-CRM 19 response (Table 31) where increased IgG2a titer to the carrier protein was seen in mice immunized with either dose of conjugate.

SUBSTITUTE SHEET rule 26 WO 99/40936 PTU9/24 PCT/US99/02847 Table 29: Anti-HibPs antibody response of mice immunized with HbOC formulated with IL-12 and AlPO4 Anti-HibPs Antibody Response (kg/mL) Vaccine Formulation Week 3 Week HbOC IL-12 AlPO 4 Pooled Pooled GMT* (ng) (jug) Serum Serum 50 100 9.73 469.16 26.92 0 100 10.04 42.55 21.30 o 0 5.12 33.19 2.25 0.1 50 100 3.18 30.95 ND o 100 4.06 15.11 ND 0 0 3.03 14.05 ND SUBSTITUTE SHEET rule 26 WO 99/40936 WO 9940936PCTIUS99/02847 -71- Table 30: Effect of IL-12 and AlPO4 on the IgG subclass response to HbOC Vaccine Formulation Anti-HibPs IgG Subclass Response at Week 5 (ELISA Endpoint Titer) HbOC IL-12 AlPO 4 IgG1 IgG2a (Psg) (ng) (Pig) 50 100 754,745 26,899 0 100 122,637 12,880 0 0 73,114 8,570 0.1 50 100 46,673 17,290 0 100 68,176 14,971 0 1 0 1 35,237 1 11,418 SUBSTITUTE SHEET rule 26 WO 99/40936 PCT/US99/02847 -72- Table 31: Anti-CRM 197 IgG response of mice immunized with HbOC formulated with IL-12 and A1P04 Vaccine Formulation Anti-CRM 197 Response at Week HbOC IL-12 A1PO 4 IgG IgG1 IgG2a IgG2b (jzg) (ng) 50 100 1,775,700 681,944 39,672 40,527 0 100 2,221,780 818,557 19,010 32,672 0 0 3,979,530 1,466,010 8,059 15,961 0.1 50 100 761,027 292,448 38,258 21,008 0 100 891,251 346,728 6,546 14,832 0 0 874,805 151,397 1,899 3,517

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. Such equivalents are intended to be encompassed by the following claims.

SUBSTITUTE SHEET rule 26

Claims

1. A vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum and optionally comprising a physiologically acceptable vehicle.

2. A vaccine composition according to claim 2, wherein the interleukin-12 is adsorbed onto the mineral suspension.

3. A vaccine composition according to claim 1 or claim 2, wherein the interleukin-12 is human interleukin-12.

4. A vaccine composition according to any one of claims 1 to 3, wherein the 0o alum is aluminum hydroxide or aluminum phosphate. A vaccine composition according to any one of claims 1 to 4, wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and combinations thereof.

6. A vaccine composition according to any one of claims 1 to 5, wherein the pneumococcal antigen is conjugated to a carrier molecule.

7. A vaccine composition according to claim 6, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.

8. A vaccine composition according to claim 7, wherein the carrier molecule is CRM1 9 7

9. A method of eliciting an immune response to a pneumococcal antigen, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a 25 physiologically acceptable vehicle. A method according to claim 9, wherein the interleukin-12 is adsorbed onto the mineral suspension.

11. A method according to claim 9 or claim 10, wherein the interleukin-12 is human interleukin-12. 30 12. A method according to any one of claims 9 to 11, wherein the alum is aluminum hydroxide or aluminum phosphate.

13. A method according to any one of claims 9 to 12, wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide R A serotypes 1, 4, 5, 6B, 9v, 14, 18C, 19F and 23F and combinations thereof. [R:\LIBUU]02496.doc:dxn

14. A method according to any of claims 9 to 13, wherein the pneumococcal antigen is conjugated to a carrier molecule. A method according to claim 14, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.

16. A method according to claim 15, wherein the carrier molecule is CRM 1 9 7

17. A method of enhancing IFN-Y response to a pneumococcal vaccine, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

18. A method of eliciting complement-fixing antibodies for a protective response to a pathogen, comprising administering to a mammalian host an effective amount of an immunogenic composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

19. An immunogenic composition comprising a mixture of a pneumococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

20. An immunogenic composition according to claim 19, wherein the interleukin- 12 is adsorbed onto the mineral suspension.

21. An immunogenic composition according to claim 21 or claim 22, wherein the interleukin-12 is human interleukin-12.

22. An immunogenic composition according to any one of claims 19 to 21, 25 wherein the alum is aluminum hydroxide or aluminum phosphate.

23. An immunogenic composition according to any one of claims 19 to 22, wherein the pneumococcal antigen is selected from the group consisting of pneumococcal capsular polysaccharide serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and combinations thereof. S 30 24. An immunogenic composition according to any one of claims 19 to 23, wherein the pneumococcal antigen is conjugated to a carrier molecule. *I •0 25. An immunogenic composition according to claim 24, whererin the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof. [R:\LIBUU]02496.doc:dxn

26. An immunogenic composition according to claim 25, wherein the carrier molecule is CRM 1 97

27. A vaccine composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a physiologically acceptable vehicle.

28. A vaccine composition according to claim 27, where the interleukin-12 is adsorbed onto the mineral suspension.

29. A vaccine composition according to claim 27 or claim 28, wherein the interleukin-12 is human interleukin-12.

30. A vaccine composition according to any one of claims 27 to 29, wherein the alum is aluminum hydroxide or aluminum phosphate.

31. A vaccine composition according to any one of claims 27 to 30, wherein the meningococcal antigen is the Neisseria meningitidis type C capsular polysaccharide.

32. A vaccine composition according to any one of claims 27 to 31, wherein the meningococcal antigen is conjugated to a carrier molecule.

33. A vaccine composition according to claim 32, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.

34. A vaccine composition according to claim 33, wherein the carrier molecule is CRM 19 7 A method of eliciting an immune response to a meningococcal antigen, comprising administering to a mammalian host an effective amount of a vaccine composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension comprising alum, and optionally comprising a 25 physiologically acceptable vehicle. *o

36. A method according to claim 35, wherein the interleukin-12 is adsorbed onto the mineral suspension.

37. A method according to claim 35 or claim 36, wherein the interleukin-12 is human interleukin-12. 30 38. A method according to claim 37, wherein the alum is aluminum hydroxide or aluminum phosphate.

39. A method according to claim 37 or claim 38, wherein the meningococcal antigen is the Neisseria meningitidis type C capsular polysaccharide.

40. A method according to any one of claims 37 to 39, wherein the meningococcal antigen is conjugated to a carrier molecule. [R:\LIBUU]02496.doc:dxn 76

41. A method according to claim 40, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.

42. A method according to claim 41, wherein the carrier molecule is CRMI 97

43. An immunogenic composition comprising a mixture of a meningococcal antigen, an adjuvant amount of interleukin-12 and a mineral suspension conprising alum, and optionally comprising a physiologically acceptable vehicle.

44. An immunogenic composition according to claim 43, wherein the interleukin- 12 is adsorbed onto the mineral suspension.

45. An immunogenic composition according to claim 43 or claim 44 wherein the interleukin-12 is human interleukin-12.

46. An immunogenic composition according to any one of claims 43 to wherein the alum is aluminum hydroxide or aluminum phosphate.

47. An immunogenic composition according to any one of claims 43 to 46, wherein the meningococcal antigen is the Neisseria meningitidis type C capsular polysaccharide.

48. An immunogenic composition according to any one of claims 43 to 47, wherein the meningococcal antigen is conjugated to a carrier molecule.

49. An immunogenic composition according to claim 48, wherein the carrier molecule is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variants thereof.

50. An immunogenic composition according to claim 49, wherein the carrier molecule is CRMI 97

51. A vaccine composition as set out in claim 1, substantially as hereinbefore 25 described with reference to any one of the Examples 1 to 6 or 8. o.

52. An immunogenic composition as set out in claim 19, substantially as hereinbefore described with reference to any one of Examples 1 to 6 or 8.

53. A vaccine composition as set out in claim 27, substantially as hereinbefore described with reference to Example 7. 30 54. An immunogenic composition as set out in claim 43, substantially as hereinbefore described with reference to Example 7.

55. A vaccine composition according to any one of claims 1 to 8 or 51, when used in a method of eliciting an immune response to a pneumococcal antigen.

56. A vaccine composition according to any one of claims 1 to 8 or 51, when used 3 in a method of enhancing IFN-y response to a pneumococcal vaccine. [R:\LIBUU]02496.doc:dxn 77

57. A vaccine composition according to any one of claims 1 to 8 or 51, when used in a method of eliciting compliment-fixing antibodies for a protective response to a pathogen.

58. A vaccine composition according to any one of claims 27 to 34 or claim 53, when used method of eliciting an immune response to a meningococcal antigen.

59. A process for producing a composition according to any one claims 1 to 8, 19 to 34, 43 to 50 or 51 to 55 substantially as hereinbefore described with reference to any one of the examples. Dated 6 February 2003 1o American Cyanamid Company Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0.o. 0* 0S *e S S* 6 S [R:\LIBUU]02496.doc:dxn