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• • 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
  • A61K39/155—Paramyxoviridae, e.g. parainfluenza 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
  • A61K39/12—Viral antigens
• • 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
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • 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
  • A61P31/12—Antivirals
• • 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
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • C—CHEMISTRY; METALLURGY
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
  • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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.
• T cells 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: Thl cells that produce IL-2 and interferon- ⁇ (IFN- ⁇ ) which augment both cellular and humoral immune responses, and Th2 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.
• IFN- ⁇ interferon- ⁇
• a substance that enhances the immunogenicity of an antigen with which it is administered is known as an adjuvant.
• certain lymphokines have been shown to have adjuvant activity, thereby enhancing the immune response to an antigen (Nencioni et_al., J. Immunol . 139 : 800-804 (1987); EP285441 to Howard et_al . ) .
• This invention pertains to vaccine compositions comprising a mixture of one or more respiratory syncytial virus (RSV) antigens, interleukin IL-12 and a mineral in suspension.
• the IL-12 may 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 RSV antigen is an RSV F and/or G protein antigen.
• the invention also pertains to methods for preparing a vaccine composition comprising mixing an RSV 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 vaccinate ' s humoral and/or cell-mediated immunity for a protective immune response, comprising administering to a vertebrate host an effective amount of a vaccine composition comprising a mixture of an RSV antigen, IL-12 and a mineral in suspension in a physiologically acceptable solution.
• the IL-12 is adsorbed onto the mineral suspension.
• Figure 1 is a graph showing the proliferative responses of splenic immunocytes from BALB/c mice vaccinated with F/AlOH plus ascending doses of IL-12. The dark shaded bars illustrate proliferation after in vi tro stimulation with native F protein.
• Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/AlOH to generate IFN- ⁇ ( Figures 2A and 2B are graphs showing the effect of IL-12 on the ability of F/
• Figure 3 is a graph showing the effects of IL-12 on the capacity of F/AlOH to induce or expand cell-mediated immune responses, and the impact of IL-12 on the capacity of F/AlOH to elicit antigen-dependent killer cells after primary immunization.
• the solid and dashed lines denote the killer cell activities observed after incubation of bronchoalveolar lavage effector cells with syngeneic RSV-infected or control target cells, respectively, 5 days after challenge.
• Figure 4 is a graph showing the effect of IL-12 on the capacity of F/AlOH to boost the cell-mediated immune responses of seropositive BALB/c mice previously infected with RSV.
• the solid and dashed lines denote the killer cell activities observed after incubation of bronchoalveolar lavage effector cells with syngeneic RSV-infected or control target cells, respectively, 5 days after challenge .
• Figure 5 is a graph illustrating the effect of recombinant IL-12 on the protective immune responses induced in BALB/c mice vaccinated with F/AlOH. The bars are one standard deviation of the geometric mean. An asterisk denotes that infectious virus was below detectable levels .
• IL-12 is produced by a variety of antigen-presenting cells, principally macrophages and monocytes . It is a critical element in the induction of Thl 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 Thl-like responses, for example, during parasitic infections, most notably Leishmaniasis (Scott et al . , U.S. Patent No. 5,571,515).
• IFN- ⁇ The effects of IL-12 are mediated by IFN- ⁇ produced by NK cells and T helper cells.
• IFN- ⁇ is critical for the induction of IgG2a antibodies to T-dependent protein antigens (Finkelman and 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).
• Interleukin-12 (IL-12) originally called natural killer cell stimulatory factor, is a heterodimeric cytokine (Kobayashi et al . , J. Exp. Med. 170 : 821 (1989).
• the expression and isolation of IL-12 protein in recombinant host cells is described in International Patent Application WO 90/05147.
• this invention pertains to vaccine compositions comprising a mixture of an RSV 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
• these vaccine compositions modulate the protective immune response to the antigen; that is, the vaccine composition is capable of eliciting the vaccinated host's cell-mediated immunity for a protective response to the pathogenic antigen.
• the antigen is the RSV F protein and/or G protein.
• 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. Further, certain T lymphocyte lines produce high levels of IL-12, thus providing a readily available source. Commercial sources of recombinant human and murine IL-12 include Genetics Institute, Inc. (Cambridge, MA).
• the antigen of this invention e.g., an RSV antigen, can be used to elicit an immune response to the antigen in a vertebrate such as a mammalian host.
• the antigen can be an RSV antigen
• RSV F protein (Collins et_al . , Proc . Natl . Acad. Sci . USA 81:7683-7687 (1984) or G protein (Satake et al . , Nuc . Acids Res . 13:7795-7812 (1985) antigen or a portion thereof which retains the ability to stimulate an immune response.
• immunogenic portions are polypeptides comprising amino acid positions 283-315, 289-315 and 294-299 of the RSV F protein. These regions include an epitope of the RSV F protein which elicits both neutralizing and antifusion antibodies (Paradiso et al . , U.S. Patent 5,639,853).
• an RSV F protein in its native dimeric form 140 kD may be used
• the method of the present invention comprises administering to a vertebrate an immunologically effective dose of a vaccine composition comprising a mixture of an antigen, e.g., an RSV antigen such as the
• an adjuvant amount of IL-12 is intended to mean a quantity of IL-12 which is sufficient to enhance or modify the immune response to the vaccine antigen, e.g., an RSV antigen such as the F and/or G protein.
• an "immunologically effective" dose of the vaccine composition is a dose which is suitable to elicit an immune response. The particular dosage will depend upon the age, weight and medical condition of the vertebrate 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' -Nbis ( 2 -hydroxyethyl -propane diamine) , methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly
• IC carbopol
• peptides e.g., muramyl dipeptide, dimethylglycine, tuftsin
• immune stimulating complexes oil emulsions
• lipopolysaccharides such as MPL® (3-0- deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Montana); and mineral gels.
• the antigens of this invention can also be incorporated into liposomes , cochleates , biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co- glycolides, or ISCOMS (immunostimulating complexes), and supplementary active ingredients may also be employed.
• the antigens of the present invention can also be administered in combination with bacterial toxins and their attenuated derivatives.
• the antigens of the invention can also be administered in combination with other lymphokines, including, but not limited to, IL-2 ,
• IL-3 IL-3
• IL-15 IFN- ⁇
• GM-CSF GM-CSF
• the vaccines can be administered to a human or animal by a variety of routes, including, but not limited to, parenteral, intrarterial, 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 RSV antigen of the present invention can be coupled to another molecule in order to modulate or enhance the immune response.
• Suitable carrier proteins include bacterial toxins which are safe for administration to mammals and immunologically effective as carriers. Examples include pertussis, diphtheria, and tetanus toxoids and non-toxic 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.
• Methods for preparing conjugates of antigens and carrier molecules are well known in the art and can be found, for example, in Dick and Burret, Contrib Microbial Immunol . 10:48-114 (Cruse JM, Lewis RE Jr, eds; Based, Krager (1989) and U.S. Patent No. 5,360,897
• the adjuvant action of IL-12 has a number of important implications.
• the adjuvanticity of IL-12 can increase the concentration of protective antibodies produced against the antigen in the vaccinated organism.
• 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. Furthermore, the adjuvant action can reduce the antigen load of a subject being immunized with a large number of vaccines in a short time.
• 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.
• IL-12 when administered with an antigen, such as an RSV antigen including, but not limited to the F protein and the G protein, and mixed with or adsorbed onto a mineral alum in suspension, can accelerate the generation of a protective immune response. This may reduce the time course of effective vaccination regimens . In some instances, it may result in the generation of a protective response in a single dose.
• the goal of the work described herein was to determine the feasibility of using recombinant IL-12 as -lo ⁇
• mice were immunized with the native fusion (F) protein of the A2 strain of RSV and ascending amounts of IL-12.
• F protein and IL-12 were adsorbed to aluminum hydroxide (AlOH, Alu-gel-STM,
• IL-12 is a powerful modifier of both systemic humoral and cell-mediated immune responses. Significant increases in complement-assisted and anti-F protein IgG2a antibody titers were observed after primary and secondary vaccination. In addition, 0.01 and 0.1 ⁇ g IL-12 had profound effects on the ability of F/AlOH to elicit cell-mediated immune responses. Five days after challenge with the A2 strain of RSV, the lungs of seronegative and seropositive mice contained augmented antigen-dependent killer cell activities.
• IL-12 increases the ability of the vaccines components to elicit immune responses governed by type 1 helper T cells (Thl) .
• Thl type 1 helper T cells
• the presence of IL-12 in the vaccine appears to be associated with increased amounts of IFN- ⁇ in the culture supernatant.
• the results imply that increased amounts of IL-12 in the formulation diminish -li ⁇
• Th2 helper T cells type 2 helper T cells
• IL-12 to alter the capacity of G/AIOH and FI-RSV to bias recipients for atypical pulmonary inflammatory responses after challenge was particularly significant.
• BAL bronchoalveolar lavage
• Thl helper T cells IFN- ⁇ - secreting Thl helper T cells.
• Thl helper T cells facilitated by IL-12 injection, hinder the ability of FI-RSV to elicit Th2 helper T cells.
• IL-12 is unable to modify the capacity of G/AIOH to predispose mice for pulmonary eosinophilia after challenge.
• the data suggest that distinct pathways exist for the control of systemic humoral immune responses and the mobilization and replication of eosinophils.
• cytokines other than IL-5 play a role in generating pulmonary eosinophilia.
• IL-12 may be present in the lungs at levels that are non-detectable in the assays employed. It is also possible that IL-12 ' s inability to limit the predilection for eosinophils results from the relatively large amounts of G protein in G/AlOH when compared to FI-RSV, or that native G protein may have several epitopes with potential to bias for eosinophilia. Some of these epitopes may be destroyed by formalin treatment. Thus, the discrepancy between FI-RSV and
• G/AIOH with respect to their capacity to bias for eosinophilia may be a quantitative phenomenon.
• the inability of IL-12 to transform the atypical pulmonary inflammatory responses predisposed by G/AIOH may be related to the uniqueness of the heavily glycosylated protein.
• IL-12 is a potent regulator of systemic humoral immune responses generated against both purified native G and F proteins of RSV. Nonetheless, the presence of IL-12 does not appear to enhance the complement fixing neutralizing antibody titers attributed to the IgG2a subclass with all RSV vaccines. Augmented serum complement-fixing neutralizing antibody titers were not observed 2 weeks after secondary immunization with either G/AIOH or FI-RSV. With respect to the FI-RSV vaccine, the failure of IL-12 to positively influence neutralizing antibody titers may reflect the destruction by formalin treatment of the F and G protein epitopes responsible for the generation of IgG2a complement fixing antibodies . As the quantity of
• IL-12 is increased in the vaccine, neutralizing and IgGl antibodies are decreased. In contrast, IL-12 reproducibly augments the IgG2a and complement enhanced neutralizing titers in response to F/AlOH (see Tables 1 and 2, below) .
• IL-12 is useful as an immune response modifier for RSV vaccines containing a mineral in suspension. Moreover, the transformation of the immune responses occurs at IL-12 doses appropriate for human use. Thus, it is clear that RSV F and G proteins, alone or in combination with other viral antigens, adjuvanted with an alum gel plus IL-12, are particularly suitable for RSV vaccine preparation.
• EXAMPLE 1 Effect of IL-12 on immunogenicity of RSV F protein adsorbed to aluminum hydroxide adjuvant
• the purpose of the study was to determine the effects of recombinant murine IL-12 on the immunogenicity of the fusion protein of RSV formulated with aluminum hydroxide adjuvant.
• Naive female BALB/c mice (8-10 weeks of age) were vaccinated intramuscularly at weeks 0 and 4 with purified native fusion (F) protein.
• the F protein was adsorbed to Alu-Gel-STM (aluminum hydroxide at 2%, Serva Fine Chemicals, Westbury, NY) .
• the vaccines were prepared such that each mouse received 3.0 ⁇ g F protein/dose, 100 ⁇ g of aluminum hydroxide (AlOH) per dose, and 0, 0.01-, 0.1, or 1.0 ⁇ g
• mice were injected with 100 ⁇ g Alu-Gel-STM in PBS alone.
• serum was collected for the determination by ELISA of geometric mean endpoint antibody titers.
• the microwells were coated with highly purified ion exchange purified F protein.
• neutralizing antibody titers were determined by the plaque reduction neutralization test in the presence and absence of complement against the A2 strain of virus.
• the results shown in Table 1 are the geometric mean endpoint antibody titers determined by ELISA.
• the neutralizing antibody titers are the geometric mean neutralizing antibody titers and were determined by the plaque reduction neutralization test in the presence (+) and absence (-) of 5% serum to supply complement.
• the antibody titers were determined 4 and 2 weeks after primary and secondary vaccination, respectively.
• IL-12 augmented systemic humoral immune responses generated by F/AlOH 4 weeks (Upper Panel, Table 1) and 2 weeks (Lower Panel, Table 1) after primary and secondary vaccination respectively.
• F/AlOH 4 weeks after primary immunization with F/AlOH formulated with either 0.1 or 1.0 ⁇ g IL-12 per dose, the total IgG endpoint antibody titers were significantly different and enhanced
• the vaccines formulated with F/AlOH plus either 0.01, 0.1 or 1.0 ⁇ g IL-12 were significantly elevated 4-, 48-, and 158-fold, respectively (Upper Panel, Table 1) . Elevations in IgG2a antibody titers were also observed 2 weeks after secondary vaccination with F/AlOH plus IL-12 (Lower Panel, Table 1) .
• mice injected with F/AlOH plus 1.0 ⁇ g IL-12 were heightened at least 7 times 4 weeks after primary immunization (Upper Panel, Table 1) .
• the neutralizing antibody titers were increased 5 and 10 times, respectively (Lower Panel, Table 1) .
• mice vaccinated with IL-12 had more potential to replicate when presented with antigen than those of mice vaccinated with aluminum gel alone (Figure 1) .
• the stimulation index of the splenic immunocytes from mice twice immunized with F/AlOH plus 0.01 ⁇ g IL-12 was nearly twice that of mice immunized with F/AlOH alone after in vi tro culture with native F protein.
• employing doses of IL-12 greater than 0.01 ⁇ g were counterproductive.
• the stimulation indices of mice vaccinated with F/AlOH plus either 0.1 or 1.0 ⁇ g IL-12 were 5-fold less.
• Table 1 The ability of recombinant murine IL-12 to modify the systemic humoral immune responses of BALB/c mice immunized with F/AlOH
• mice were vaccinated intramuscularly on weeks 0 and 4 with native F protein (3 ⁇ g/dose) adsorbed to aluminum hydroxide (AlOH, 100 ⁇ g/dose) .
• IL-12 was added to the vaccines at the indicated doses.
• Control mice were injected with PBS plus AlOH.
• the upper and lower panels depict antibody titers 4 and 2 weeks after primary and secondary vaccination respectively.
• the numbers are the geometric mean endpoint antibody titers determined by ELISA.
• t The numbers are the geometric mean neutralizing antibody titers and were determined by the plaque reduction neutralization test in the presence (+) or absence (-) of 5% complement. There were 5 mice per group .
• c P ⁇ 0.05 vs.
• EXAMPLE 2 The effect of administration of IL-12 at a distal site on the ability of F/AlOH to induce systemic humoral immune responses .
• mice were injected IM with F/AIOH in one thigh and received 10-fold ascending doses of IL-12 in the contralateral thigh.
• groups of mice were injected once with a vaccine composed of F/AIOH formulated with 1 of 3 10-fold ascending doses of IL-12. The vaccine was incubated overnight at 4°C to allow maximum time for adsorption of IL-12 to AlOH. Additional control mice were vaccinated either with F/AIOH alone, F protein in PBS alone, or PBS plus AlOH alone.
• sera were collected for the determination of geometric mean endpoint anti-F protein total and subclass IgG antibody titers by ELISA.
• Table 2 The results depicted in Table 2 confirmed the capacity of IL-12 to augment the systemic humoral immune responses induced after vaccination with F/AIOH.
• the anti-F protein total IgG antibody titers elicited by F/AIOH plus either 10 or 100 ng IL-12 were significantly greater 4 weeks after primary immunization.
• the presence of either 10 or 100 ng IL-12 in the vaccines was associated with statistically enhanced protein specific IgG2a antibody titers (Table 2) .
• Table 2 the data also suggested that in a single dose protocol, IL-12 must be present at the local site of injection.
• mice When mice were primed with F/AlOH alone and IL-12 was injected at a distal site, statistically lower anti-F protein antibody titers were obtained (Table 2) .
• the protein specific IgG2a antibody titers of mice injected with F/AIOH plus 100 ng IL-12 were 10 times greater than those of cohort mice immunized with F/AIOH alone plus 100 ng IL-12 administered in the contralateral thigh.
• t Naive female BALB/c mice were primed intramuscularly (IM) with native fusion (F) protein (3 ⁇ g/dose) adsorbed to aluminum hydroxide adjuvant (AlOH) .
• F/AIOH was administered in combination with 10-fold ascending doses of recombinant murine IL-12 (100, 10, 1 ng IL-12/dose) .
• Two immunization strategies with F/AIOH plus IL-12 were employed: 2 inj .
• mice which were injected IM with F/AIOH in one thigh and received 10-fold ascending doses of IL-12 in the contralateral thigh; 1 inj., 1 site indicates that mice were injected once with a vaccine composed of F/AlOH plus 1 of 3 10-fold ascending doses of IL-12. Additional control mice were vaccinated either with F/AIOH alone, F protein in PBS alone, or PBS plus AlOH alone. The numbers are geometric endpoint titers of 5 mice per group.
• mice were vaccinated with F/AIOH alone, or were intranasally administered mock infected Hep2 cell lysate. Four weeks after primary vaccination the mice were challenged intranasally with RSV A2 ( ⁇ 10 6 PFU) and bronchoalveolar lavage (BAL) was performed 5 days later. The cytolytic capacity of the inflammatory cells was determined directly in a standard 4-hour 51 Cr release assay after incubation with syngeneic RSV-infected and control target cells.
• mice vaccinated with F/AIOH plus 0.01 ⁇ g IL-12 did not lyse syngeneic control targets (dashed lines) not infected with virus.
• EXAMPLE 4 The effect of IL-12 on the ability of F/AlOH to induce cell-mediated immune responses in seropositive recipients.
• IL-12 on the capacity of F/AIOH to expand cell-mediated immune responses in recipients previously infected with RSV.
• Naive female BALB/c mice (8-10 weeks of age) were primed by experimental infection with the A2 strain of RSV. Four weeks later, the mice were injected intramuscularly with one of three F protein based vaccines plus recombinant murine IL-12.
• the vaccines were composed of F protein (3 ⁇ g/dose) adsorbed to aluminum hydroxide (AlOH, 100 ⁇ g/dose) plus one of three 10-fold ascending doses of IL-12 (0.01, 0.1, and 1.0 ⁇ g IL-12 /dose) .
• AlOH aluminum hydroxide
• the aluminum hydroxide was prepared at Wyeth-Lederle Vaccines and Pedatrics .
• mice were primed by infection with RSV and secondarily vaccinated with F/AIOH alone, or were intranasally administered mock infected Hep2 cell lysate. Two weeks after secondary vaccination the mice were challenged intranasally with RSV A2 ( ⁇ 10 6 PFU) and bronchoalveolar lavage (BAL) was performed 5 days later. The cytolytic capacity of the inflammatory cells was determined directly in a standard 4-hour 51 Cr release assay after incubation with syngeneic RSV-infected and control target cells .
• Naive female BALB/c mice (8-10 weeks of age) were vaccinated intramuscularly (IM) at weeks 0 and 4 with either 1 ⁇ g purified native attachment glycoprotein (G) adsorbed to aluminum hydroxide (AlOH, Alu-gel-STM, Serva, 100 ⁇ g dose) adjuvant or 0.1 ml formalin-inactivated RSV (FI-RSV) .
• the FI-RSV vaccine was a facsimile of the original Lot-100 vaccine formulated by Pfizer and was adsorbed to AlOH (1600 ⁇ g dose) . This vaccine was used as a benchmark for atypical pulmonary inflammatory response, an undesirable immune response for subunit vaccines .
• IL-12 was added to G/AIOH and FI-RSV in 10-fold ascending doses (0.1 to 10.0 ⁇ g IL-12/dose). Additional companion groups of mice were infected (0.05 ml intranasally) with the A2 strain of RSV, injected IM with 0.1 ml formalin-inactivated parainfluenza virus type 3 (FI-PIV3) vaccine adsorbed to AlOH (1600 ⁇ g/dose), or were intranasally administered 50 ⁇ l of mock-infected Hep2 cell lysate (MOCK) . Two weeks after secondary vaccination, serum was collected for the determination of geometric mean endpoint antibody titers by ELISA in microwells coated with either affinity-purified G protein or ion exchange purified F protein. Geometric mean neutralizing antibody titers were also determined by the plaque reduction neutralization test in the presence and absence of complement against the A2 strain of the virus.
• BAL bronchoalveolar lavage
• the ratio of serum anti-F protein IgGl to IgG2a antibody titers observed after secondary vaccination with FI-RSV was 75.3 (Table 4).
• the ratio of serum anti-G protein IgGl to IgG2a antibody titers observed 2 weeks after secondary vaccination was greater than 184.2 (Table 3).
• results in the “% EOS” column are the geometric mean relative percentage of eosinophils (EOS) enumerated in the BAL fluids 5 days after challenge with virus.
• EOS eosinophils
• ND denotes not determined.
• IL-5 was detected by capture ELISA and quantified from a standard curve.
• results in the “IL-5 (OD) " column are the geometric mean optical density (OD 490 ) .
• mice vaccinated with FI-RSV 5 days after challenge was significantly elevated when compared with that of control mice vaccinated with FI-PIV3 (6.5%) and undergoing primary infection, or mice immunized by experimental infection ( ⁇ 1.0%) (Table 6).
• mice vaccinated with FI-RSV 106 pg/ml
• the amount of IL-5 secreted into the lavage fluids of mice vaccinated with FI-RSV was significantly elevated when contrasted with those fluids from recipients of either FI-PIV3 ( ⁇ 8 pg/ml) or infectious virus ( ⁇ 35 pg/ml) 5 days after infection (Table 6) .
• the G protein employed in this study contained concentrations of F protein that were immunogenic for BALB/c mice.
• Noteworthy were the serum anti-F protein total IgG antibody titers observed 2 weeks after secondary vaccination with G/AIOH (Table 4) .
• the data implied that both G and the contaminating F protein in G/AIOH, like the FI-RSV vaccine, induced primarily Th2 helper T cell subsets.
• the serum anti-F protein IgGl to IgG2a antibody ratios after secondary vaccination were greater than 617 (Table 4) .
• the serum G protein-specific IgGl to IgG2a antibody ratios after secondary vaccination with G/AIOH were greater than 1251 (Table 3) .
• mice vaccinated with G/AlOH 167 pg/ml
• the amount of IL-5 secreted into the lavage fluids of mice vaccinated with G/AlOH was significantly elevated when contrasted with those fluids from recipients of either mock infected Hep-2 cell lysates ( ⁇ 35 pg/ml) , infectious virus ( ⁇ 35 pg/ml) , or FI-PIV3 ( ⁇ 8 pg/ml) 5 days after infection (Table 6) .
• F protein dependent type 1 helper T cell responses.
• the systemic humoral immune responses elicited by the F protein contained in infectious virus was characterized by secondary serum anti-F protein IgGl to IgG2a antibody ratios less than 1.0 (Table 4).
• atypical pulmonary inflammatory responses were not associated with previous RSV infection (Table 6) . Pulmonary eosinophilia was not observed in naive mice undergoing primary infection (Table 6) .
• IL-12 had a profound impact on the humoral immune responses generated after vaccination with either the facsimile vaccine or G/AlOH.
• the inhibitory effect of IL-12 was also observed following addition of 10 ⁇ g to G/AIOH.
• IL-12 limited the ability of G/AIOH to generate anti-F protein IgGl antibody titers.
• secondary immunization with the G/AlOH plus 10 ⁇ g IL-12 generated IgGl antibody titers that were 5-fold and significantly less (Table 4) .
• the serum antibody titers were 17 fold less than those of mice twice immunized with FI-RSV plus 0.1 ⁇ g IL-12 (Table 4).
• Tables 3 and 4 are the geometric mean endpoint antibody titers determined by ELISA.
• l/2a is the ratio of geometric mean IgGl to IgG2a subclass antibody titers.
• NT denotes not tested;
• ND denotes not determined.
• the addition of IL-12 did not appear to dramatically alter the magnitude of anti-G protein total IgG antibody titers generated after secondary vaccination with either FI-RSV or G/AIOH (Table 3). However, the presence of IL-12 in the vaccines significantly diminished IgGl antibody titers, while the IgG2a protein specific antibody titers were significantly elevated (Table 3).
• IgG2a antibody subclass titers elicited after secondary vaccination with G/AIOH plus 1.0 ⁇ g IL-12 were 1,300 times greater when compared to those generated by G/AIOH alone (Table 4) .
• immunization with G/AIOH plus 10 ⁇ g IL12 resulted in F protein-specific IgG2a subclass antibody titers that were comparable to those generated after vaccination with G/AIOH plus 1.0 ⁇ g IL-12.
• IL-12 has the ability, via the induction of distinct helper T cell subsets, to modify the infiltration and/or replication of eosinophils in the pulmonary tissues after challenge. This was exemplified by the effect of IL-12 on the capacity of the vaccines to predispose mice for increased amounts of IL-5 and relative percentages eosinophils in the lungs 5 days after challenge (Table 6) .
• Table 6 the addition of 0.1 or 1.0 ⁇ g IL-12 to the FI-RSV vaccine significantly reduced the amount of IL-5 and relative number of eosinophils, respectively.
• IL-12 did not appear to have any transforming effect on the capacity of G/AIOH to predispose BALB/c mice to pulmonary eosinophilia after challenge (Table 6) . It was noteworthy that atypical pulmonary inflammatory responses were not observed in mice immunized by infection with the A2 strain of virus. Furthermore, IL-5 and eosinophilia were not observed in the lungs of control mice 5 days after primary infection (Table 6) . IFN- ⁇ was at or near baseline for all groups examined (data not shown) .
• Table 5 The results shown in Table 5 are the geometric mean neutralizing antibody titers .
• the titers were determined 4 and 2 weeks after primary and secondary vaccination respectively by the plaque reduction neutralization test and in the presence (+) or absence (-) of 5% serum as a source of complement.
• FI-RSV plus 10 ⁇ g IL-12 The presence of 10 ⁇ g IL-12 in G/AlOH was also associated with a statistically significant 10-fold reduction in complement-independent neutralizing antibodies 2 weeks after secondary vaccination. However, the magnitude of the complement-dependent neutralizing antibody titers elicited by the F protein contaminated G/AIOH were equivalent to that of mice vaccinated by experimental infection (Table 5) .
• mice b P ⁇ 0.05 vs. total serum IgG antibody titers from mice vaccinated with FI-RSV alone or plus 0.1 or 1.0 ⁇ g L-12.
• c P ⁇ 0.05 vs. total serum IgG antibody titers from mice vaccinated with FI-RSV plus 0.1 ⁇ g IL-12.
• d P ⁇ 0.05 vs. serum IgGl antibody titers from mice vaccinated with FI-RSV plus 1.0 or 10.0 ⁇ g IL-12.
• Vaccine IL-12 ( ⁇ g) IgG IgGl IgG2a l/2a
• mice vaccinated with FI-RSV plus 0.1 or 1.0 ⁇ g IL-12 There were 5 mice per group.
• b P ⁇ 0.05 vs. total serum IgG antibodies from mice vaccinated with FI-RSV plus 0.1 or 1.0 ⁇ g IL-12.
• c P ⁇ 0.05 vs. total serum IgG antibodies from mice vaccinated with FI-RSV plus 0.1, 1.0, or 10.0 ⁇ g IL-12.
• VACCINE IL-12 ( ⁇ g) (+) (-) (+) (-)
• the numbers are the geometric mean neutralizing antibody titers.
• the titers were determined 4 and 2 weeks after primary and secondary vaccination respectively by the plaque reduction neutralization test and in the presence (+) or absence (-) of 5% complement. There were 5 mice per group.
• mice were immunized intramuscularly with either FI-RSV or G/AlOH alone, or plus 10-fold ascending doses of recombinant murine IL-12.
• mice were immunized intramuscularly on weeks 0 and 4 with formalin-inactivated RSV (FI-RSV) plus 10 fold ascending doses of recombinant murine IL-12.
• Control mice were vaccinated with formalin-inactivated parainfluenza virus type 3 (FI-PIV3), natural fusion protein admixed with QS-21 (F/QS-21) or infected with the A2 strain of RSV. Additional control mice received an intranasal administration of mock-infected Hep2 cell lysate (MOCK) or were injected intramuscularly with PBS/QS-21.
• FI-RSV formalin-inactivated RSV
• FI-PIV3 formalin-inactivated parainfluenza virus type 3
• QS-21 natural fusion protein admixed with QS-21
• Additional control mice received an intranasal administration of mock-infected Hep2 cell lysate (MOCK) or were injected intramuscularly with
• the numbers are the geometric mean relative percentage of eosinophils (EOS) enumerated in the BAL fluids 5 days after challenge with virus. ND denotes not determined. c IL-5 was detected by capture ELISA and quantified from a standard curve. The numbers are the geometric mean optical density (OD 490 ) . d P ⁇ 0.05 vs. the eosinophils detected in mice vaccinated with either FI-PIV3 or FI-RSV plus 1.0 ⁇ g
• EXAMPLE 6 The Effect of IL-12 on the Capacity of F/AlOH to Generate Protective Immune Responses in BALB/c Mice.
• Additional control mice were immunized by experimental infection with the A2 strain of RSV.
• All mice were challenged with RSV A2 (50 ⁇ l, ⁇ 5 X 10 6 PFU) .
• the level of virus replication in the pulmonary tissues was assessed four days later. Briefly, the lung and tracheal tissues were removed en bloc, homogenized, clarified, snap frozen, and stored at -70°C until assayed for infectious virus.
• the level of virus replication in the respiratory tract tissues was assessed in a plaque assay employing Hep-2 cell monolayers .
• Sera were also collected four weeks after primary vaccination for the determination of geometric mean endpoint anti-F protein total and subclass IgG antibody titers by ELISA. Geometric mean serum neutralizing antibody titers were also revealed by the plaque reduction neutralization test against the A2 strain of virus in the presence or absence of 5% complement.
• the results shown in Figure 5 are "the geometric mean plaque forming units (PFU) of virus per gram of pulmonary tissue determined four days after challenge.
• the data depicted in Table 7 are the geometric mean endpoint anti-F protein IgG antibody titers determined by ELISA.
• the neutralizing antibody titers are the geometric mean neutralizing antibody titers and are determined by the plaque reduction neutralization test in the presence (+) or absence (-) of 5% complement. The antibody titers were determined four weeks after primary vaccination.
• mice primed with F/AlOH plus either 10 or 100 ng IL-12 and challenged with RSV A2 contained greater than 3 log 10 PFU virus .
• Antigen IL-12 (ng) IgG IgGl IgG2a + -
• mice were primed intramuscularly (IM) with ion exchange purified F protein (30 ng/dose) adsorbed to aluminum hydroxide adjuvant (AlOH, 100 ⁇ g/dose) adjuvant.
• the F/AlOH was administered with PBS alone, or in combination with 100 or 10 ng recombinant murine IL- 12/dose.
• Additional control mice were immunized by experimental infection with the A2 strain of RSV.
• the numbers are geometric endpoint IgG and neutralizing antibody titers (log 10 ) ⁇ 1 standard deviation of the mean of 5 mice per group.
• the neutralizing antibody titers (log 10 ) were determined in the presence (+) or absence (-) of 5% complement.

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