Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/095—Neisseria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
  • A61K39/092—Streptococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
  • A61K2039/55527—Interleukins
  • A61K2039/55538—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

• the immune system uses many mechanisms for attacking pathogens; however, not all of these mechanisms are necessarily activated after immunization.
• Protective immunity induced by vaccination is dependent on the capacity of the vaccine to elicit the appropriate immune response to resist or eliminate the pathogen. Depending on the pathogen, this may require a cell- mediated and/or humoral immune response.
• 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 ⁇ (IFN- ⁇ ) which augment both cellular and humoral immune responses, and TH-2 cells that produce IL-4, IL-5 and IL-10 which augment humoral immune responses (Mosmann et al . , J. Immunol . 126: 234,8 (1986)).
• an adjuvant A substance that enhances the immunogenicity of an antigen with which it is administered is known as an adjuvant.
• lymphokines have been shown to have adjuvant activity, thereby enhancing the immune response to an antigen (Nencioni et al . , J. Immunol . 139:800-804 (1987); EP285441 to Howard et al . ) .
• This invention pertains to vaccine compositions comprising a mixture of one or more pneumococcal or meningococcal antigens, the interleukin IL-12 and a mineral in suspension.
• the IL-12 can be either adsorbed onto the mineral suspension or simply mixed therewith.
• the IL-12 is adsorbed onto a mineral suspension such as alum (e.g. , aluminum hydroxide or aluminum phosphate) .
• alum e.g. , aluminum hydroxide or aluminum phosphate
• 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 .
• pneumococcal polysaccharide serotypes are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F, (Pnl, Pn4, Pn5, Pn6B, Pn9V, Pnl4, Pnl8C, Pnl9F, Pn23F)
• the meningococcal polysaccharide is type C (Men C) .
• 1,000 ng increased the IgGl, IgG2a, IgG2b and IgG3 response to alum-adsorbed Pnl4 or Pn ⁇ B. In addition they increased the IgG2a response to Pn4 and Pn9V.
• 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.
• the IL-12 is adsorbed onto the mineral suspension.
• the invention also pertains to methods for eliciting or increasing a vaccinee ' s IFN- ⁇ -producing T cells and complement-fixing IgG antibodies for a protective immune response, comprising administering to a mammalian, e.g., human or primate, host an effective amount of a vaccine composition comprising a mixture of antigen, IL-12 and a mineral in suspension in a physiologically acceptable solution.
• the IL-12 is adsorbed onto the mineral suspension.
• PnPs-14-CRM 197 vaccine comprises a serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant of diphtheria toxoid
• CRM 197 cross-reacting material
• PnPs ⁇ B- CRM 197 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.
• MPL® 3-O-deacylated monophosphoryl lipid A
• RIBI ImmunoChem Research, Inc. Hamitton, Montana
• IL-12 is produced by a variety of antigen- presenting cells, principally macrophages and monocytes. It is a critical element in the induction of TH-1 cells from naive T cells. Production of IL-12 or the ability to respond to it has been shown to be critical in the development of protective TH-1-like responses, for example, during parasitic infections, most notably Leishmaniasis (Scott et al . , U.S. Patent No. 5,571,515). The effects of IL-12 are mediated by IFN- ⁇ produced by NK cells and T helper cells. Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor, is a heterodimeric cytokine (Kobayashi et al . , J. Exp . Med. 170 : 827 (1989)). The expression and isolation of IL-12 protein in recombinant host cells is described in International Patent Application WO 90/05147, published May 17, 1990.
• this invention pertains to vaccine compositions comprising a mixture of such an antigen, IL-12 and a mineral in suspension.
• the IL-12 is adsorbed onto a mineral suspension such as alum (e.g., aluminum hydroxide or aluminum phosphate) .
• alum e.g., aluminum hydroxide or aluminum phosphate
• 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.
• a 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.
• 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.
• a meningococcal antigen particularly a meningococcal polysaccharide
• the meningococcal antigen is optionally conjugated to a carrier molecule, such as in a meningococcal glycoconjugate.
• Type C Neisseria meningi tidis is exemplified herein; however, this type is not to be construed to limit the scope of the invention, as other types are also suitable for use herein.
• IL-12 can be obtained from several suitable sources . It can be produced by recombinant DNA methodology; for example, the gene encoding human IL-12 has been cloned and expressed in host systems, permitting the production of large quantities of pure human IL-12. Also
• the antigen of this invention can be used to elicit an immune response to an antigen in a mammalian host.
• the antigen can be a serotype 14 or 6B pneumococcal polysaccharide or a portion thereof which retains the ability to stimulate an immune response.
• Additional suitable antigens include polysaccharides from other encapsulated bacteria and conjugates thereof, secreted toxins and outer membrane proteins .
• the method comprises administering to the mammal, e.g., human or primate, an immunologically effective dose of a vaccine composition comprising a mixture of an antigen, such as a pneumococcal antigen or a pneumococcal conjugate, and an adjuvant amount of IL-12 adsorbed onto a mineral in suspension.
• an "immunologically effective" dose of the vaccine composition is a dose which is suitable to elicit an immune response.
• the particular dosage of IL-12 and the antigen will depend upon the age, weight and medical condition of the mammal to be treated, as well as on the method of administration. Suitable doses will be readily determined by the skilled artisan.
• the vaccine composition can be optionally administered in a pharmaceutically or physiologically acceptable vehicle, such as physiological saline or ethanol polyols such as glycerol or propylene glycol .
• the vaccine composition may optionally comprise additional adjuvants such as vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamin, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N,N- dicoctadecyl-N' -N'bis (2-hydroxyethyl-propane diamine) , methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dime hylglycine, tuftsin; immune stimulating complexes; oil emulsions; liposaccharides such as MPL® and mineral gels.
• surface active substances
• the antigens of this invention can also be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly- lactide-co-glycolides , or ISCOMS (immunostimulating complexes) , and supplementary active ingredients may also be employed.
• the antigens of the present invention can also be administered in combination with bacterial toxins and their attenuated derivatives .
• the antigens of the present invention can also be administered in combination with other lymphokines, including, but not limited to, IL-2, IL-3 , IL-15, IFN- ⁇ 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.
• the IL-12 and the antigen will be co-administered; however, in some instances the skilled artisan will appreciate that the IL-12 can be administered close in time but prior to or after vaccination with the antigen.
• the pneumococcal and meningococcal antigens of the present invention can be coupled to a carrier molecule in order to modulate or enhance the immune response.
• Suitable carrier proteins include bacterial toxins rendered safe by chemical or genetic means for administration to mammals and immunologically effective as carriers. Examples include pertussis, diphtheria, and tetanus toxoids and non- oxic mutant proteins (cross-reacting materials (CRM) ) , such as the non-toxic variant of diphtheria toxoid, CRM 197 .
• Fragments of the native toxins or toxoids, which contain at least one T- cell epitope, are also useful as carriers for antigens, as are outer membrane protein complexes.
• 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.
• vaccination regimens call for the administration of antigen over a period of weeks or months in order to stimulate a "protective" immune response.
• a protective immune response is an immune response sufficient to protect the immunized organism from productive infection by a particular pathogen or pathogens to which the vaccine is directed.
• alum-formulated vaccine comprising IL-12 adsorbed onto AlP0 4 and a serotype 14 or serotype 6B pneumococcal polysaccharide conjugated to CRM 197 , which normally induces a response dominated by IgGl
• 0.2 ⁇ g of IL-12 substantially increased the IgG2a and IgG3 subclasses in both Balb/c and Swiss Webster mice, but had little or no effect on IgGl.
• Enhancement of IgG2b to Pnl4 was seen with Swiss Webster mice; 0.2 ⁇ g of IL-12 had the same effect as 25 ⁇ g 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.
• the opsonophagocytic activity of the antisera for Pnl4 pneumococci from mice receiving 0.2 ⁇ g 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®.
• IgG2a and IgG2b antibodies are very efficient at activating the complement system, whereas IgGl antibodies are not.
• the complement system consists of a series of plasma proteins which come together around IgG2a or IgG2b bound to antigen (e.g., bacteria) to form a large molecular complex. Deposition of this complex on the surface of bacteria results in the killing of the bacteria by perforating the cell membrane (bactericidal activity) or by facilitating the recognition of the bacteria by phagocytic cells (such as polymorphonuclear cells (PMN) used in this study) , which take up the bacteria and kill them (opsonophagocytosis) .
• phagocytic cells such as polymorphonuclear cells (PMN) used in this study
• IFN- ⁇ is critical for the induction of IgG2a antibodies to T-dependent protein antigens (Finkelman and Holmes, Annu . Rev. Immunol . S:303-33 (1990)) and IgG3 responses to T-independent antigens (Snapper et al . , J. Exp . Med. 175:1367-1371 (1992)).
• 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) .
• high doses of IL-12 severely inhibited the IgGl response of Swiss Webster mice. Whether this is associated with decreased production of TH-2 cytokines after the second vaccination is unclear.
• IL-12 exhibited either only immunomodulatory activity or behaved both as a "classical" adjuvant, and a immunomodulator, depending on the vaccine.
• 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.
• IL-12 enhanced the IgG2a response to CRM 197 , which is consistent with its ability to favor the induction of TH-1-like helper cells (IFN- ⁇ producers) .
• IL-12 also enhanced the IgGl response to CRM 197 after primary and secondary vaccination.
• IgGl antibodies are normally associated with TH-2-like helper cells which produce IL-4.
• Inclusion of 0.1 ⁇ g IL-12 into an AlP0 4 -based Pnl8C conjugate vaccine (which on its own induced a 10-fold higher CRM 197 response) had no effect on IgGl but substantially increased the IgG2a titer.
• the IgG2a titer achieved with 0.1 ⁇ g IL-12 was at least as high as that obtained with 5 ⁇ g IL-12 in the absence of A1P0 4 . It should be noted, however, that the presence of A1P0 4 does not preclude the enhancement of IgGl responses by IL-12. In mice immunized with the Pnl4 conjugate on A1P0 4 , a 0.2 ⁇ g dose of IL-12 enhanced the IgGl, IgG2a and IgG2b titers to CRM 197 .
• the differences in the effect on IgGl may reflect differences in the immunogenicity of the two conjugates for CRM 197 IgG responses; the Pnl4 conjugate on A1P0 4 induced 10-fold lower CRM 197 IgG titers so that there was room for IL-12 to enhance an IgGl response, but not when mice were immunized with Pnl8C conjugate on AlP0 4 .
• the fact that MPL® and QS-21TM markedly increased the IgGl titers in mice immunized with Pnl8C conjugate on A1P0 4 indicates that the IgGl response had not been maximally stimulated.
• the nature of the saccharides on the conjugates may be a factor.
• IL-12 probably exerts its adjuvant effect differently than MPL® or QS-21TM.
• IL-12 markedly enhanced the CRM 197 IgG2a titers in mice immunized with Pnl8C conjugate but had minimal effects on IgG2b.
• MPL® and QS-21TM enhanced the titers of both IgG subclasses.
• IgG2b is induced by cytokines other than, or in addition to, the IFN- ⁇ that drives switching to IgG2a and is known to mediate the immunomodulatory effects of IL-12.
• TGFb 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 ⁇ g IL-12 caused IgG2a and IgG2b to be elevated to similar levels which were equivalent to the titers promoted by 25 ⁇ g MPL®.
• 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 A1P0 4 .
• IL- 12 /A1P0 4 enhanced both the IgGl and IgG2a subclasses to Pn6B, indicating that the apparent lack of enhancement of the Pnl4 IgGl response by IL-12 is probably not a generalizable phenomenon.
• This work further supports the idea that the mechanisms of adjuvant activity by IL- 12 and MPL® are not equivalent. Both adjuvants enhanced the Pn6B IgGl and IgG2a titers to similar levels, but MPL® was more effective at promoting IgG2b and IgG3 antibodies.
• IL-12/AlP0 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 ⁇ g dose of PnPs-14-CRM 197 glycoconjugate, i.e., 10-fold higher than in the Pn6B studies .
• the applicability of IL-12 to more complex pneumococcal vaccines was demonstrated using a nonavalent vaccine containing glycoconjugates from serotype 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F pneumococci.
• IL-12 with A1P0 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 197 ) which is poorly immunogenic in mice.
• IL-12 was tested with a glycoconjugate vaccine against type C Neiserria meningitidis (MenC) and a glycoconjugate vaccine against type B Hemophilus influenzae (HbOC) .
• MenC Neiserria meningitidis
• HbOC glycoconjugate vaccine against type B Hemophilus influenzae
• Formulating that vaccine with 50 ng IL-12 and A1P0 4 enhanced the IgG2a titers to MenC capsular polysaccharide although not to HbOC.
• A1P0 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 AlP0 4 , thereby enhancing its persistence in the animal; additional studies indicate that IL-12 rapidly binds to alum (data not shown) .
• the local inflammatory effect of A1P0 4 may induce cytokines that potentiate the biological activity of IL-12.
• PnPsl4-CRM 197 is a conjugate of capsular polysaccharide from serotype 14 pneumococci covalently linked to the genetically detoxified diphtheria toxin, CRM 197 , by reductive amination.
• MPL® 3-0- deacylated monophosphoryl lipid A, RIBI Immunochem Research, Inc., Hamilton Montana
• the vaccinations were given subcutaneously three weeks apart .
• Sera were collected at week 3 (primary response) and weeks 5 and 7 (secondary responses 2 and 4 weeks after boosting) .
• the sera were analyzed for IgG antibodies to PnPs-14.
• the sera were also analyzed for the ability to promote opsonophagocytic killing of type-14 pneumococci by human polymorphonuclear cells (PMN) .
• Type 14 pneumococci were opsonized with dilutions of antisera and C8-depleted serum as a source of complement. They were then incubated with human polymorphonuclear cells (PMN) , and the percent of bacteria surviving was determined by colony counts .
• Table 1 shows that 1 ⁇ g and 5 ⁇ g IL-12 substantially reduced the anti-PnPs-14 IgG response in mice immunized with conjugate formulated with AlP0 4 .
• 0.2 ⁇ g IL-12 induced substantially higher IgG2a, IgG2b and IgG3 titers but left the IgGl levels essentially unaltered.
• the IgG subclass profile induced by 0.2 ⁇ g IL-12 was indistinguishable from that obtained with 25 ⁇ g MPL®, and sera from mice receiving these adjuvants had higher opsonophagocytic activity than those from mice immunized with a vaccine containing only A1P0 4 (Table 2) .
• the higher doses of IL-12 markedly reduced the IgGl antibodies; at 5 ⁇ g cytokine, IgGl titers were at least 10-fold lower than in mice immunized without IL-12. This effect was apparent both during the primary response and after boosting.
• IgG2b showed the greatest reduction such that vaccines containing 1 ⁇ g or 5 ⁇ g 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 ⁇ g IL-12, after the second vaccination these subclasses were higher than in the controls .
• IgG subclass response to a PnPsl4-CRM 197 conjugate vaccine formulated with A1P0 4 A 0.2 ⁇ g dose of IL-12 increased the IgG2a, IgG2b and IgG3 response to Pnl4 without affecting the IgGl response. Higher doses of IL-12 resulted in a marked reduction in the IgGl and IgG2b titers. IgG2a and IgG3 titers also appeared to decline at these doses, but they were still higher than in mice immunized in the absence of IL-12.
• Example 2 demonstrates that the IgG subclass changes were associated with enhanced induction of IFN- ⁇ -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-1-like.
• Table 1 Effect of IL-12 on the immunogenicity of PnPs- 14-CRM 197 /alum vaccine
• EXAMPLE 2 Nature of T helper cells induced by
• Pneumococcal conjugate vaccine (PnPs-14- CRM 197 /A1P0 4 ) formulated with IL-12
• mice were bled and reimmunized with the same vaccine formulation used in the first immunization.
• week 5 the mice were bled once more.
• week later their draining lymph node cells and splenocytes were harvested and cultured for six days with CRM 197 , lysozyme, ConA or in medium alone.
• Culture supernatants from parallel cultures were harvested at day 3 and day 6 and assayed for IFN- ⁇ , IL-5 and IL-10 by ELISA.
• lymph node cells from mice immunized without IL-12 produced IFN- ⁇ , IL-5 and IL-10 when stimulated with CRM 197 in vi tro (Table 4) .
• Adding IL-12 to the vaccine dramatically increased the antigen-specific production of IFN- ⁇ and abolished the ability of the lymphoid cells to produce IL-5 and IL-10.
• Maximal IFN- ⁇ production was obtained with the lowest dose of IL-12 (0.2 ⁇ g) ; higher doses, particularly 5 ⁇ g, appeared to reduce the levels of this cytokine. This was most clearly seen in cultures stimulated with 1 ⁇ g/mL CRM 197 .
• the reduction in IFN- ⁇ with higher doses of IL-12 may not reflect a generalized suppressive phenomenon since IFN- ⁇ production in response to Con A was the same regardless of the dose of IL-12 in the vaccine.
• lymph node cells and splenocytes from mice immunized with vaccine containing IL-12 continued to produce elevated levels of IFN- ⁇ in response to stimulation with CRM 197 compared to mice immunized without IL-12 (Table 5) .
• 0.2 ⁇ g to 1.0 ⁇ g IL-12 were optimal doses of IL-12 for augmentation of an IFN- ⁇ response.
• IL-5 and IL-10 production were differentially affected.
• the 1.0 and 5.0 ⁇ g doses of IL-12 essentially eliminated the IL-5 response but, by comparison, had only a minor effect on IL-10 production.
• IL-12 (5.0 ⁇ g) abolished the ability of splenocytes but not lymph node cells to produce IL-10 (Tables 5 and 6) .
• Lysozyme 30 ⁇ 0.22 ⁇ 0.22 ⁇ 0.22 ⁇ 0.22 ⁇ 0.22
• Lysozyme 30 ⁇ 0.14 0.21 0.21 0.21
• Lysozyme 30 ⁇ 0.4 ⁇ 0.4 ⁇ 0.3 ⁇ 0.3
• Lysozyme 30 ⁇ 0.3 ⁇ 0.3 ⁇ 0.2 ⁇ 0.2
• Lysozyme 30 ⁇ 0.2 ⁇ 0.2 ⁇ 0.3 ⁇ 0.2
• IFN- ⁇ CRM 30 9.8 86.9 58.7 62.0
• Lysozyme 30 ⁇ 0.4 ⁇ 0.4 ⁇ 0.3 ⁇ 0.3
• Lysozyme 30 ⁇ 0.3 ⁇ 0.3 ⁇ 0.2 ⁇ 0.2
• Lysozyme 30 ⁇ 0.2 ⁇ 0.2 ⁇ 0.2 ⁇ 0.2 ⁇ 0.2
• mice (10 per group) were immunized with 1 ⁇ g Pnl8C conjugate formulated with or without 100 ⁇ g A1P0 4 .
• the vaccines were supplemented with either IL- 12 (0.2, 1 or 5 ⁇ g) , 100 ⁇ g MPL® or 20 ⁇ g QS-21TM.
• Normal mouse serum (0.5% final) was used to stabilize the diluted IL-12 and was added to all vaccines, regardless of composition.
• 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) .
• the Pnl8C IgG responses are presented in Table 7.
• a dose of 5 ⁇ g of IL-12 caused a 3 -fold rise in the IgG titer of pooled week 5 sera, whereas vaccine formulated with 1 ⁇ g of IL-12 appeared to induce no Pnl8C response.
• the lowest dose of IL-12 (0.1 ⁇ g) induced the same response as the A1P0 4 -formulated vaccine not containing IL-12.
• the vaccine formulated with MPL®/AlP0 4 induced the highest frequency of responses; 7/10 mice gave OD>0.2, in contrast to QS-21TM/AlP0 4 and
• A1P0 4 alone each of which induced 4/10 responders.
• IgG2b titers promoted by IL-12 were 20-fold lower than those induced by MPL®.
• 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.
• IL-12 can act as adjuvant for IgG responses to a protein antigen.
• the increase in both IgGl and IgG2a titers suggest that, within this model at least, IL-12 enhances priming of both TH-1-like and TH-2-like helper cells by PnOsl8C-CRM 197 conjugate in the absence of A1P0 4 .
• Adjuvant ( ⁇ g/dose) Wk3 Wk5 IgGl IgG2a IgG2b IgG3
• Table 8 Effect of IL-12 on CRM 197 IgG response three weeks after vaccination with PnOsl ⁇ C conjugate o O ⁇ > I o t
• EXAMPLE 4 Effect of IL-12 on the IgG response of Swiss Webster mice to bivalent vaccine containing PnPs6B-CRM 197 and PnPs-14-CRM 197
• mice were immunized subcutaneously at weeks 0 and 3 with a vaccine comprising 0.1 ⁇ g per dose of PnPs6B-CRM 197 glycoconjugate (a conjugate of capsular polysaccharide from serotype 6B pneumococci covalently linked to CRM 197 ) plus 0.1 ⁇ g per dose of PnPsl4-CRM 197 glycoconjugate.
• the vaccines were administered with 0, 8, 40, or 200 ng IL-12, either alone or in combination with 100 ⁇ g alum (A1P0 4 ) .
• Normal mouse serum (0.25%) was included as a carrier protein to stabilize the IL-12 at low concentrations.
• mice A control group of mice was immunized with the vaccine formulated with 100 ⁇ g monophosphoryl lipid A (MPL®) . 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.
• MPL® monophosphoryl lipid A
• 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 A1P0 4 .
• 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 A1P0 4 (no IL-12) .
• formulating the vaccines with 8-200 ng of IL-12 in the absence of A1P0 4 did not alter the IgG subclass response.
• these doses of IL-12 when combined with A1P0 4 resulted in substantially increased IgGl 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 A1P0 4 ; however, these titers appeared to be substantially lower than those induced by vaccine formulated with MPL®.
• A1P0 4 plus 40 ng IL-12 induced a significantly higher Pn6B titer than vaccine formulated without adjuvant.
• 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 A1P0 4 alone.
• 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 A1P0 4 , did not enhance the response to PnPsl4 after primary or secondary vaccination.
• subclass analysis indicated that IL-12 did not enhance the IgG2a titers when formulated with IL-12.
• 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.
• EXAMPLE 5 Comparison of the effect of IL-12 in the presence or absence of alum on the murine immune response to monovalent PnPsl4- CRM 197 conjugate vaccine
• mice (8 per group) were immunized subcutaneously at week 0 with 1 ⁇ g PnPsl4-CRM 197 conjugate formulated with or without 100 ⁇ g A1P0 4 and either no IL-12 or with 8, 40, 200, 1,000 or 5,000 ng IL-12.
• Normal mouse serum (0.25%) was included as a carrier protein to stabilize IL-12 at low concentrations.
• 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.
• 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) .
• mice receiving 5,000 ng IL-12, but not lower doses of IL-12, in the absence of A1P0 4 had significantly higher spleen weights than those receiving vaccine containing neither alum nor IL-12 (Table 14) .
• Vaccines containing A1P0 4 induced higher spleen weights when formulated with 40 to 5000 ng IL-12.
• the data indicate that formulating IL-12 with AlP0 4 greatly enhanced a biological activity of the cytokine, i.e., its ability to cause increased spleen weight one week after vaccination.
• IL-12 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 AlP0 4 or was formulated without A1P0 4 (Table 18) . In the absence of A1P0 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 AlP0 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.
• IgG2b and IgG3 titers were assayed on pooled sera only (Table 15) .
• the IgG response to CRM 197 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) .
• 40 ng IL-12 appeared to modestly increase the IgG titers to
• CRM 197 after two vaccinations.
• the highest IgG titers to CRM 197 were obtained when the vaccine was formulated with both A1P0 4 and 8-40 ng IL-12.
• the heightened adjuvant activity of IL-12 co-formulated with AlP0 4 is indicated by the finding that, on their own, 40 ng IL-12 and A1P0 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 IgGl response to CRM 197 regardless of whether the vaccine was formulated with or without A1P0 4 (Tables 19 and 20) .
• IL-12 substantially increased the week 5 IgG2a titers to CRM 197 after immunization with vaccines containing AlP0 4 (Table 19) . Again the optimal dose of IL-12 appeared to be 40 ng. The cytokine appeared to increase the IgG2b titers induced by vaccine containing A1P0 4 .
• Cytokine production by spleen cells taken two weeks after secondary vaccination (week 5) revealed effects of IL-12 on the priming of both IFN- ⁇ and IL-5 producing cells.
• Splenocytes from mice immunized in the absence of A1P0 4 and IL-12 produced detectable levels of IL-5, but not IFN- ⁇ , when stimulated with CRM 197 in vi tro
• Table 14 Spleen weights of Balb/c mice one week after subcutaneous immunization with 1 ⁇ g PnPsl4-CRM 197 conjugate formulated with or without 100 ⁇ g A1P0 4 and the indicated doses of IL-12
• Adjuvant CRM 197 IgG Titer IgG Subclasses at
• Table 20 IgGl titers to CRM 197 in Balb/c mice immunized with PnPsl4-CRM 197 conjugate formulated with IL-12 and A1P0,
• Table 21 Cytokine production by splenocytes from mice immunized twice with PnPsl4- ⁇ CRM 197 formulated with IL-12 in the presence and absence of AlP0 4 o
• EXAMPLE 6 Effect of IL-12 /AlP0 4 on the humoral response to a nonavalent pneumococcal glycoconjugate vaccine
• Formulating the nonavalent vaccine with A1P04 enhanced the IgG response to several serotypes including PnPs4, PnPs ⁇ B, PnPs9V and PnPsl4, especially when the lowest dose of conjugate (0.1 ⁇ g) was used (Tables 24-27) .
• Addition of IL-12 did not appear to further enhance the IgG response to these serotypes .
• addition of 50 or 1,000 ng IL-12 to 5 ⁇ g of vaccine containing A1P04 resulted in higher geometric mean IgG titers to this serotype and higher proportion of mice with PnPsl ⁇ C IgG titers above 10,000 (Table 23). The responses to PnPsl, 5, 19F and 23F were not evaluated.
• 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 ⁇ g) or with A1P0 4 plus IL-12.
• Sera from the week 5 bleed were analyzed for IgG antibodies to CRM 197 .
• Table 23 Effect of IL-12 on the response to PnPsl ⁇ C in mice immunized with 5 ⁇ g nonavalent pneumococcal glycoconjugate vaccine formulated with A1P04
• 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 ⁇ g) or with A1P0 4 plus IL-12.
• Individual mouse sera from the week 5 bleed were analyzed for IgG antibodies to PnPsl ⁇ C.
• 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 ⁇ g) or with A1P04 plus IL-12.
• Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs4.
• Table 25 Effect of IL-12 on the PnPs6B response of mice immunized with nonavalent pneumococcal glycoconjugate vaccine formulated with A1P04
• 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 ⁇ g) or with A1P04 plus IL-12.
• Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs ⁇ B.
• Table 26 Effect of IL-12 on the PnPs9V response of mice immunized with nonavalent pneumococcal glyconconjugate vaccine formulated with A1P04
• 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 ⁇ g) or with a A1P04 plus IL-12.
• Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs9V.
• Table 27 Effect of IL-12 on the PnPsl4 response of mice immunized with nonavalent pneumococcal glyconconjugate vaccine formulated with A1P04
• 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 ⁇ g) or with A1P04 plus IL-12.
• Sera from the week 5 bleed were analyzed for IgG antibodies to PnPsl4.
• EXAMPLE 7 The effect of IL-12 and A1P04 on the immune response to Neiserria meningi tidis type C (menC) glyconconjugate vaccine
• mice Neiserria meningi tidis type C (menC) .
• Swiss Webster mice were immunized at weeks 0 and 3 with 0.1 ⁇ g or 1 ⁇ g of MenC glycoconjugate formulated alone, with A1P0 4 (100 ⁇ g) 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.
• Example 8 The effect of IL-12 and A1P04 on the immune response to Hemophilus influenzae type b glyconconjugate vaccine (HbOC)
• Hemophilus influenzae type b Swiss Webster mice (10 per group) were immunized at weeks 0 and 3 with 0.1 ⁇ g or 1.0 ⁇ g of glyconconjugate vaccine consisting of capsular polysaccharide from Hemophilus influenzae type b (HibPs) conjugated to CRM 197 .
• the vaccine HbOC
• A1P04 100 ⁇ g
• A1P04 100 ⁇ g
• A1P04 100 ⁇ g
• a mixture of IL-12 50 ng
• A1P04 Normal mouse serum was not added to the vaccine.
• the mice were bled at weeks 3 and 5.
• the antibody response to HibPs was measured using a Farr assay which measures all antibodies binding to the saccharide regardless of isotype, i.e., IgM, IgG and IgA.
• the IgG subclass response was measured by ELISA. Additionally, the IgG and IgG subclass response to CRM 197 was also determined by ELISA.
• mice immunized with vaccine formulated alone, with A1P04 or IL-12 plus A1P04 regardless of the dose of conjugate used for immunization (Table 29).
• mice immunized with 1 ⁇ g 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 30) .
• analysis of individual mouse sera showed that this was due to a single mouse having a titer of approximately 10,000 ⁇ g/mL.
• 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.
• IL-12 and A1P04 appeared to enhance the IgG2a titer 3 -fold in mice immunized with 1 ⁇ g of conjugate. However, this was no different than the titer obtained with vaccine adjuvanted with A1P04 alone. In mice immunized with 0.1 ⁇ g 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 197 response (Table 31) where increased IgG2a titer to the carrier protein was seen in mice immunized with either dose of conjugate.

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