WO2024124052A1 - Immune adjuvants for polysaccharide vaccines - Google Patents

Immune adjuvants for polysaccharide vaccines Download PDF

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Publication number
WO2024124052A1
WO2024124052A1 PCT/US2023/082976 US2023082976W WO2024124052A1 WO 2024124052 A1 WO2024124052 A1 WO 2024124052A1 US 2023082976 W US2023082976 W US 2023082976W WO 2024124052 A1 WO2024124052 A1 WO 2024124052A1
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turbo
vips
polysaccharide
mice
cells
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PCT/US2023/082976
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French (fr)
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Kishore R. ALUGUPALLI
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Thomas Jefferson University
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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

Definitions

  • Vaccination is a cornerstone strategy for inducing broad protective immunity against a range of viral and bacterial pathogens in both animal and human subjects. While vaccines focusing on protein targets are well established, vaccines targeting polysaccharide or polysaccharide/protein conjugates have also been the subject of research interest.
  • One reason for the attractiveness of this strategy is the fact that many infectious agents, particularly bacteria and some enveloped viruses, express surface polysaccharides that play key roles in host immune evasion and pathogenesis. As such, directing host antibody immune responses against these targets would act to block target cell infection and mark bacterial cells and viral particles for subsequent immune system -mediated clearance.
  • polysaccharides by themselves are poorly immunogenic. Even, even when combined with conjugated proteins and/or traditional, human-compatible adjuvants, polysaccharides sometimes fail to generate long-term antibody and T cell responses, despite repeated booster immunizations.
  • the present disclosure relates to immunogenic compositions useful for inducing immunity to polysaccharide antigens and/or and polysaccharide-containing antigens. Also included are methods of stimulating an immune response in subjects in need thereof and methods of treating, ameliorating, and/or preventing diseases and/or disorders in subjects comprising administering the immunogenic compositions of the disclosure.
  • the present invention provides an immunogenic composition
  • an immunogenic composition comprising: one or more antigenic molecules between 0.01 pg/ml and 50.0 pg/ml; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or a derivative thereof.
  • MPLA Monophosphoryl Lipid A
  • Di-acyl lipopeptide e.g. Pam2 CSK4
  • the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • the polysorbate 80 or an analogue or derivative thereof is between 0.1% and 10% (w/w).
  • the antigenic molecule is selected from the group consisting of a polysaccharide, a protein, a peptide, a peptide/poly saccharide conjugate, a protein/polysaccharide conjugate and any combination thereof.
  • the antigenic molecule is derived from a bacterium.
  • the antigenic molecule is derived from a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite.
  • the present invention provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more antigenic molecules; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
  • MPLA Monophosphoryl Lipid A
  • D-acyl lipopeptide e.g. Pam2 CSK4 or
  • the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • the polysorbate 80 is between 0.1% and 10% (w/w).
  • the antigenic molecule is selected from the group consisting of a polysaccharide, a peptide, and a peptide/polysaccharide conjugate.
  • the antigenic molecule is derived from a bacterium.
  • the antigenic molecule is derived from a virus.
  • the immune response is independent of T cells. In certain embodiments, the immune response is dependent on T cells. In certain embodiments, the immune response is mediated by B cells.
  • B cells are stimulated by B cell receptor and toll-like receptor (TLR) signaling.
  • TLR toll-like receptor
  • the B cells are Bib cells or Bib equivalent cells.
  • the immune response comprises the generation of antigenspecific antibodies.
  • the antibodies comprise IgM antibodies, IgG antibodies, or a combination of IgM and IgG antibodies.
  • the immune response generates long-term immune memory.
  • the subject is human, primate, bovine, porcine, ovine, canine, feline or murine.
  • the current disclosure provides a kit comprising the immunogenic composition of any one of claims 1-6.
  • the current disclosure provides a method of treating, ameliorating, and/or preventing a disease in subject in need thereof, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein.
  • the disease is associated with the antigenic molecule.
  • the disease is a polysaccharide encapsulated bacterial infection.
  • the polysaccharide antigen is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiriti dis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptococcus, Pseudomonas aeruginosa, Klebsiella, Pasteurella, Brucella, Francisella, Helicobacter, Vibrio and Bacillus anthracis.
  • the bacterial infection is a non-polysaccharide encapsulated bacterial infection.
  • the non-polysaccharide antigen is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Streptococcus, Chamydia, Escherichia coli, Meningococcal group B, Staphylococcus aureus, and Group A and B Streptococcus.
  • FIG. l is a table illustrating the various B cell subsets in mice.
  • FIG. 2 illustrates that a long term Bib cell expansion is concurrent with the resolution of bacteremia in mice deficient in Btk, a kinase required for BCR-mediated signal: Involvement of a co-stimulatory signal.
  • FIG. 3 is a diagram illustrating B cell receptor (BCR) signaling, highlighting the role of Btk, a kinase for signal transduction.
  • BCR B cell receptor
  • FIG. 4 is a diagram illustrating Toll-Like Receptor (TLR) signaling highlighting the role of MyD88, an adaptor for signal transduction of multiple members of the TLR family.
  • FIG. 5 is a diagram illustrating the critical role for the PH domain of Btk for docking to the membrane and kinase domains of Btk for the autophosphorylation, that is essential for Btk’s function.
  • TLR Toll-Like Receptor
  • FIGs. 6A-6B illustrate that MyD88 and Btk mediated signaling are essential for T cellindependent pathogen-specific IgM immune responses.
  • One of the mouse strains has a substitution mutation in PH domain (i.e., R28C) and the other one has a mutation/deletion in the kinase domain of the Btk protein.
  • FIG. 6A illustrates the levels of IgM after stimulation in indicated mouse lines.
  • FIG. 6B illustrates bacterial cells present in peripheral blood in wildtype or double knockout (DKO, deficient in both Btk and Myd88) mice.
  • DKO double knockout
  • FIGs. 7A-7B illustrate that plain polysaccharide vaccines induce poor and short-lived antibody response in adults.
  • FIG. 7A is a table comparing seropositivity rates (%) and geometric mean titers (GMTs) of antigen-specific antibody during the first 12 months after vaccination with two indicated polysaccharide vaccines.
  • FIG. 7B displays similar data in graph form.
  • FIGs. 8A-8H illustrate that polysaccharide conjugated to a widely used carrier proteins such as CRM197 for the production of conjugate vaccines does not induce efficient response in Typhoid and invasive pneumococcal disease endemic countries.
  • Graphs show GMC, as measured by ELISA in adults in Pakistan (FIG. 8A), India (FIG. 8B), children in Pakistan (FIG. 8C) and India (FIG. 8D), older infants in Pakistan (FIG. 8E) and India (FIG. 8F), and infants in Pakistan (FIG. 8G) and India (FIG.8H).
  • FIG. 9 is a table illustrating that Alum-based adjuvants induce a modest ( ⁇ 2-fold) increase in antibody response to Vi PS conjugate vaccines.
  • FIG. 10 is a diagram illustrating that the ViPS antigen is isolated from bacteria. Therefore, the ViPS preparation is contaminated with bacterial components such as LPS.
  • ViPS from Sanofi Pasteur was isolated from S. Typhi strain Ty2
  • ViPS from US FDA was isolated from Citrobacter freundii strain WR7011.
  • ViPS from International Vaccine Institute, South Korea was isolated from S. Typhi, clinical isolate C6524.
  • Mouse peritoneal macrophages were incubated with various concentrations of ViPS for three sources or LPS (from Sigma Aldrich as a positive control). The levels of IL-6 as a readout were measured by ELISA.
  • FIGs. 11A-11C show that mice deficient in TLR4, which recognizes LPS are impaired in responding to plain ViPS vaccine (obtained from US FDA, lot 5) as well as World Health Organization (WHO) pre-approved ViPS-Tetanus Toxoid conjugate vaccine (ViPS-TT, commercially known as TypBar TCV from Bharat Biotech, India). This suggests that LPS present in these vaccines plays a significant role in antibody responses.
  • WHO World Health Organization
  • FIGs. 12A-12B illustrate that Phenol extracted ViPS is poorly immunogenic. However, its immunogenicity can be promoted by Turbo, which refers to a non-limiting liposome adjuvant of the disclosure.
  • ViPS obtained from US FDA
  • C57BL6 mice were immunized i.m. with original stock ViPS (2.5 pg) or phenol-extracted ViPS (2.5 pg) with and without Turbo (5 pg), and levels of ViPS-specific (FIG. 12 A) IgM and (FIG. 12B) IgG were measured by ELISA.
  • FIG. 13 are diagrams comparing the structures of bacterially derived Lipid A (top) and two synthetic Lipid A molecules (bottom).
  • FIGs. 14A-14C are diagrams of the components of the immune adjuvant of the current disclosure.
  • FIG. 14A illustrates monophosphroyl Lipid A (MPLA), l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC), and polyoxyethylene sorbitan monooleate (Polysorbate 80).
  • FIG. 14B is a diagram of an example liposome comprised of the composition of the disclosure.
  • FIG. 14C is a chart of liposome size as compared to concentration of particles.
  • D IO The portion of particles with diameters smaller than this value is 10%.
  • D50 The portions of particles with diameters smaller and larger than this value are 50%. Also known as the median diameter.
  • D90 The portion of particles with diameters below this value is 90%.
  • FIG. 15 is a diagram of a typical immunization strategy for a mouse study using the Turbo adjuvant of the disclosure with various immunogens.
  • FIGs. 16A-16B FIG. 16A illustrate that Turbo driven IgM and IgG isotype responses are independent of mouse background. C57BL6, 129Sv or BALB/c mice were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo (5 pg) and levels of ViPS-specific IgM and IgG were measured by ELISA. FIG. 16B illustrate that Turbo also enhances IgM and IgG isotype responses to ViPS-TT conjugate vaccine.
  • FIGs. 17A-17B illustrate that Turbo also enhances IgM and IgG isotype responses to 4-Hydroxy-3 -nitrophenylacetyl (NP) hapten conjugated to Ficoll (NP-Ficoll), a high molecular weight synthetic polysaccharide widely used for studying T cell-independent B cell activation.
  • FIG. 17B illustrate that Turbo also enhances IgM and IgG isotype responses to NP conjugated to Chicken Gamma Globulin (NP-CGG), a model antigen for studying T cell- dependent B cell responses.
  • NP-Ficoll 4-Hydroxy-3 -nitrophenylacetyl
  • NP-Ficoll hapten conjugated to Ficoll
  • FIG. 17B illustrate that Turbo also enhances IgM and IgG isotype responses to NP conjugated to Chicken Gamma Globulin (NP-CGG), a model antigen for studying T cell- dependent B cell responses.
  • FIGs. 18A-18B illustrate that a single immunization of ViPS with Turbo induces long- lasting and sustained IgG response increases protective immunity in young (3 -week-old) mice.
  • C57BL6 mice (3-week-old) were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo (5 pg) and levels of ViPS-specific IgM and IgG were measured by ELISA.
  • Statistical differences were determined using Two-way ANOVA with Bonferroni post-test. ** denotes p ⁇ 0.01. (FIG 18B).
  • mice were infected i.p. with 3 x 10 4 CFUs of ViPS expressing S.
  • FIG. 19 illustrate that Turbo promotes IgG response in infant mice.
  • Ten-day old C57BL6 mice were immunized i.m. with ViPS-TT conjugate vaccine (1.0 pg) alone, admixed with Turbo (2.5 pg), and levels of ViPS-specific IgG was measured by ELISA.
  • FIGs. 20A-20B illustrate the use of Turbo vaccination in mice with and without a squalene-based adjuvant (SE).
  • C57BL6 mice (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) alone, admixed with Turbo (5 pg), 2% Squalene-emulsion (SE) or both, and levels of ViPS-specific IgM (FIG. 20A) and IgG (FIG. 20B) were measured by ELISA.
  • Statistical differences were determined using Two-way ANOVA. **** p ⁇ 0.001, **p ⁇ 0.01 and N.S. denotes not significant.
  • FIG 21 illustrates the comparison of various Turbo formulations.
  • the structures of all the three TLR4 agonists are shown in Figure 13.
  • C57BL/6 mice (8-12-week-old) were immunized i.m. with ViPS vaccine (2.0 pg) alone, admixed with Turbo (5 pg), and levels of ViPS-specific IgM and IgG isotypes were measured by ELISA.
  • FIGs. 22A-22B illustrate the comparison of various Turbo formulations and other adjuvants namely alum and squalene-based adjuvant (SE).
  • C57BL/6 mice (8-12-week-old) were immunized i.m. with ViPS-TT conjugate vaccine (2.0 pg) alone, admixed with Turbo (5 pg), 2% Squalene-emulsion (SE) or Alum at 0.5 mg (0.25 mg aluminum hydroxide & 0.25 mg magnesium hydroxide), and levels of ViPS-specific IgM (FTG. 22A) and IgG (FIG. 22B) were measured by ELISA.
  • FIGs. 23A-23B illustrate that Turbo-driven antibody responses are not dependent on NLRP3 inflammasome or pyropotosis.
  • Wildtype or mice deficient in NLPR3, Caspl, or GsdmD on C57BL6 background (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) admixed with Turbo (5 pg), and levels of ViPS-specific IgM (FIG. 23 A) and IgG (FIG. 23B) were measured by ELISA.
  • FIGs. 24A-24B illustrate that Turbo-driven antibody responses is dependent on TLR4- MyD88 axis.
  • Wildtype or mice deficient in TLR4, or TLR adaptor proteins MyD88 or Trif on C57BL6 background (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) admixed with Turbo (5 pg), and levels of ViPS-specific IgM (FIG. 24A) and IgG (FIG. 24B) were measured by ELISA.
  • ViPS 2.5 pg
  • FIG. 24B IgG
  • Statistical differences were determined using Two-way ANOVA. **** p ⁇ 0.001, **p ⁇ 0.01.
  • FIGs. 25A-25B illustrate that Turbo induces increased primary and secondary anti-ViPS IgG response, suggesting that turbo promotes IgM and IgG memory B cells. Mice were reimmunized 62 days after primary immunization to measure the booster response.
  • FIGs. 26A-26B illustrate the induction of anti-ViPS IgG response under antigen-limiting conditions by Turbo.
  • C57BL6 mice were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo at various concentrations (FIG. 26A); or with ViPS at various concentrations + Turbo at 5 pg (FIG. 26B).
  • ViPS-specific IgG was measured by ELISA.
  • FIGs. 27A-27B illustrate that Turbo vl & v2 (without and with 2% Tween 80, respectively) induces increased anti-ViPS IgG response.
  • FIG. 28 is a schematic of a proposed model illustrating the integration of various molecular signals and cellular interactions.
  • FIG. 29 is a table illustrating that many approved polysaccharide subunit vaccines lack adjuvants.
  • FIGs. 30A-30C are graphs that illustrate that Turbo, when used as an adjuvant, promotes a rapid and robust antibody response in infant (10 days old) mice.
  • the vaccine comprised ViPS-TT (TypBar TCV) conjugate as an antigen.
  • FIG. 30A illustrates the IgM (top) and IgG (bottom) levels in each individual animal at the indicated time post-immunization.
  • FIG. 30B illustrates the same data in graphical form and at timepoints out to 120 days.
  • FIG. 30C illustrates the levels of RC60 in blood, liver, and spleen of vaccinated animal after challenge.
  • FIG. 31 are graphs that illustrate that Turbo promotes a sustained antibody response in adult (2 - 4 months old) mice when vaccinated with an unconjugated ViPS vaccine. Graphs indicate IgM (left) and IgG (right) levels in peripheral blood at the indicated timepoints.
  • FIG. 32 are graphs that illustrate that Turbo adjuvant promotes all IgG isotypes in adult mice vaccinated with a ViPS-TT (TypBar TCV) conjugate vaccine. Two mouse strains were used, C57BL/6J (top) and 129Sl/SvImJ (bottom).
  • FIG. 33 are graphs that illustrate that Turbo adjuvant promotes a rapid and robust antibody response in aged mice (1.6 years old) to ViPS-TT (TypBar TCV) conjugate vaccine.
  • FIG. 34 are graphs that illustrate that Turbo promotes heightened recall responses to booster immunization, indicating the generation of B cell memory.
  • FIGs. 35A-35B are graphs that illustrate that Turbo adjuvant promotes long-lasting antibody responses, which indicates the generation of antigen-specific, long-lived plasma cells. Graphs indicate the comparison of Turbo (filled black dots) with alum (open red dots).
  • FIG. 36 are graphs that illustrate that Turbo promotes a rapid and robust antibody response in infant (10 days old) mice challenged with hapten-conjugated antigens.
  • FIGs. 37A-37B are graphs that illustrate that a wide range of TLR4 agonists combine with Turbo adjuvant to generate significant immunity to both conjugated and unconjugated ViPS vaccines.
  • FIG. 37A depicts unconjugated ViPS antigen.
  • FIG. 37B depicts a ViPS-tetanus toxoid conjugate vaccine.
  • FIGs. 38A-38B are graphs that show that Turbo adjuvant function is independent of NLRP3 inflammasome or pyroptosis mechanisms.
  • FIG. 38A illustrates data in various knockout mice using unconjugated ViPS as the immunogen.
  • FIG. 38B illustrates data in the same mice using phenol extracted ViPS antigen.
  • FIG. 39 are graphs that illustrate that Turbo adjuvant engages TLR4-MyD88, TLR4-Trif, and Caspase 11 signaling axes when administered with a ViPS-TT conjugate vaccine.
  • FIGs. 40A-40B are graphs that illustrate that conventional dendritic cells (eDCs) are not required for Turbo adjuvant function.
  • FIG. 40A depicts the use of an unconjugated ViPS antigen.
  • FIG. 40B depicts use of a conjugated ViPS-tetanus toxoid vaccine.
  • FIGs. 41 A-41B are graphs that illustrate that macrophages but not neutrophils are required for the adjuvant effects of Turbo.
  • FIG. 41A illustrates use of an unconjugated ViPS antigen.
  • FIG. 42B illustrates use of a tetanus toxoid conjugated vaccine.
  • FIG. 42 is a schematic of a macrophage-centric model for Turbo adjuvant function.
  • FIG. 43 are graphs that illustrate that Turbo enhances serotype-specific IgM responses to meningococcal polysaccharide conjugate vaccines Menveo and MenQuadfi.
  • FIG. 44 are graphs that illustrate that Turbo enhances serotype-specific IgG responses to meningococcal polysaccharide conjugate vaccines Menveo and MenQuadfi.
  • FIG. 45 are graphs that illustrate that Turbo adjuvanticity to ViPS conjugate vaccine is Trif pathway dependent, and admixing Turbo to the vaccine is required for the optimal antibody response.
  • FIG. 46 are graphs that illustrate that Turbo enhances antibody response to unconjugated ViPS vaccine (Typhim Vi) in a TLR4 and Myd88 dependent manner.
  • FIG. 47 are graphs that illustrate that Turbo enhances antibody response to unconjugated ViPS vaccine in both inbred and outbred mice.
  • FIG. 48 are graphs that illustrate that Turbo enhances antibody response to conjugated ViPS vaccine (ViPS-TT) in both inbred and outbred mice.
  • ViPS-specific IgM, IgGl, IgG2b, IgG2c, and IgG3 levels were measured by ELISA.
  • FIGs. 51A-51C illustrate the elimination of immunostimulatory components in the ViPS preparation results in the loss of immunogenicity.
  • FIG. 50B Peritoneal exudate cells were incubated with indicated stimulants for 24 h and IL-6 levels in the supernatant was measured by ELISA.
  • FIG. 50C Wildtype mice were immunized i.m.
  • ViPS-specific IgM and IgG levels were measured by ELISA.
  • FIGs. 52A-52B illustrate Turbo adjuvanticity under antigen-limiting conditions.
  • FIG. 53 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to phenol extracted ViPS in both inbred and outbred mice.
  • FIG. 54 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to ViPS subunit vaccines in both inbred and outbred mice.
  • FIG. 55 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to ViPS conjugate vaccine in both inbred and outbred mice using Typbar TCV® vaccines.
  • FIGs. 56A-56B illustrate that Turbo adjuvanticity to unconjugated ViPS vaccine is independent of sex. Male mice are represented in FIG. 56A, while female mice are represented in FIG. 56B.
  • FIGs. 57A-57B illustrate that Turbo adjuvanticity to conjugated ViPS vaccine is independent of sex.
  • Male mice are represented in FIG. 57A.
  • FIGs. 58A-58D illustrate Turbo as an adjuvant promotes Vi PS-specific antibody responses to unconjugated Vi PS in the young mice and enhances Vi PS-specific antibody responses to conjugated Vi PS vaccine (Typbar TCV®) in adult, and aged (old) mice.
  • FIG. 59 illustrates that the immunogenicity of Meningococcal subunit vaccines is dependent on MyD88, an adaptor for multiple TLR signaling.
  • Wildtype (C57BL/6J) male and female mice of indicated genotype were immunized i.m with 50 pl of (FIG. 59A) Menveoi® (4 micrograms of MenACWY) or (FIG. 59B) MenQuadfi® (2.5 micrograms of MenACWY) or with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA).
  • Meningococcal serotype-specific IgM and IgG levels were measured by ELISA.
  • FIGS. 60A-60B illustrate that a prime-boost strategy with Turbo as an adjuvant enhances antigen-specific response in infant mice and 100% seroconversion.
  • Wildtype (C57BL/6J) male and female mice of 9 days (infant) were immunized subcutaneously with 25 microliters of (FIG. 60A) Menveo® (2 micrograms of MenACWY) or (FIG. 60B) MenQuadfi® (1 .25 micrograms of MenACWY) or with (Black filled circles) and without (white circles) Turbo (containing 2.5 pg of MPLA).
  • Meningococcal serotype-specific IgM and IgG levels were measured by ELISA.
  • FIGs. 61A-61C illustrate that Turbo as an adjuvant promotes antibody responses to multivalent meningococcal vaccines in young adult and aged mice.
  • Meningococcal serotype-specific IgM and IgG levels were measured by ELISA.
  • FIG. 62 illustrates that Turbo as an adjuvant promotes antibody responses to H. influenzae PRP-conjugate vaccines Hiberix.
  • FIG. 63 illustrates that Turbo as an adjuvant promotes antibody responses to Rabies viral vaccine, Imovax.
  • FIGs. 64A-64D illustrate a comparison of Turbo adjuvanticity with that of Squalene- based emulsion or Alum in the context of bacterial or synthetic polysaccharide, Protein conjugated polysaccharide vaccine or protein antigen.
  • Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with various bacterial polysaccharide (ViPS), protein conjugated- Vi PS vaccine (Typbar TCV), NP-CGG, a commonly used model of protein antigen, or a synthetic polysaccharide NP-Ficoll.
  • ViPS bacterial polysaccharide
  • Typbar TCV protein conjugated- Vi PS vaccine
  • NP-CGG protein conjugated- Vi PS vaccine
  • FIG. 64A-Ficoll a synthetic polysaccharide
  • FIG. 64A 2.5 pg of unconjugated Vi PS with indicated adjuvants (Turbo containing 5 pg of MPLA) or 5% squalene emulsion.
  • FIG. 64B Mice immunized with 2.5 pg protein conjugated- Vi PS vaccine (Typbar TCV) adjuvanted with Turbo, Squalene-based emulsion, or Alum (ImjectTM Alum Adjuvant at containing 1 mg aluminum hydroxide & 1 mg magnesium hydroxide),
  • FIG. 64C NP-CGG adjuvanted with Turbo or Alum;
  • FIG. 64D NP-Ficoll adjuvanted with and without Turbo.
  • ViPS-specific antibody (A&B) and NP-specific antibody (C&D) levels were measured by ELISA.
  • FIGs. 65A-65D illustrate that Turbo as an adjuvant induces durable antibody responses, promotes antibody cl ass- switching to all four IgG isotypes and affinity maturation, and enhanced germinal centers in the draining lymph nodes.
  • mice were boosted with the same antigens in the same area of primary immunization.
  • NP-specific antibody levels were measured by ELISA.
  • the ELISA values obtained with NP30-BSA or NP-18 BSA coated plates measures NP-specific antibodies of diverse affinities, whereas the ELISA values obtained with NP-2 BSA coated plates measures high affinity antibodies.
  • the ratio of NP2/NP18 ELISA values indicate affinity maturation.
  • FIG. 65A Turbo-adjuvanted protein antigen shows durable IgG responses of all 4 isotypes.
  • FIG. 65B Turbo promotes affinity maturation of all 4 IgG subclasses.
  • FIGs. 65C & 65D Represents the size of germinal centers (GC).
  • FIGs. 65A-65D illustrate that Turbo as an adjuvant induces durable antibody responses, promotes antibody cl ass- switching to all four IgG isotypes and affinity maturation, and enhanced germinal centers in the draining lymph nodes.
  • mice were boosted with the same antigens in the same area of primary immunization.
  • NP-specific antibody levels were measured by ELISA.
  • the ELISA values obtained with NP30-BSA or NP-18 BSA coated plates measures NP-specific antibodies of diverse affinities, whereas the ELISA values obtained with NP-2 BSA coated plates measures high affinity antibodies.
  • the ratio of NP2/NP18 ELISA values indicate affinity maturation.
  • FIG. 65A Turbo-adjuvanted protein antigen shows durable IgG responses of all 4 isotypes.
  • FIG. 65B Turbo promotes affinity maturation of all 4 IgG subclasses.
  • FIGs. 65C & 65D Represents the size of germinal centers (GC).
  • FIG. 66A-66B illustrate that Turbo adjuvanticity occurs when it is administered ipsilaterally to the antigen.
  • Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the right thigh of the hind limb with NP-CGG, a commonly used model of protein antigen, or protein conjugated-Vi PS vaccine (Typbar TCV) were bedside mixed without (diamond symbol) or Turbo (Black circles; ipsilateral).
  • NP-specific antibody FIG. 66A-66B illustrate that Turbo adjuvanticity occurs when it is administered ipsilaterally to the antigen.
  • Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the right thigh of the hind limb with NP-CGG, a commonly used model of protein antigen, or protein conjugated-Vi PS vaccine (Typbar TCV) were bedside mixed without (diamond symbol) or Turbo (Black circles;
  • ViPS-specific antibody (FIG. 66B) levels were measured by ELISA.
  • FIGs. 67A-67B illustrate that Turbo adjuvanticity to unconjugated polysaccharide vaccines is dependent on TLR4 and MyD88.
  • FIG. 68 illustrates that Turbo-mediated adjuvanticity for polysaccharide vaccines is dependent on co-stimulatory molecules CD40, CD80 and CD86.
  • Wildtype (C57BL/6J) male and female mice of indicated genotype were immunized i.m with 50 pl of phenol extracted ViPS (2.5 micrograms) admixed with Turbo (containing 5 ig of MPLA).
  • ViPS-specific IgM and IgG isotype levels were measured by ELISA.
  • FIG. 69 illustrates the structures of the TLR4, TLR1/2, and TLR2/6 homodimerizing agonists MPLA, Pan CSIGi, and ParrnC SK i, respectively.
  • FIG. 70 illustrates the structural basis of lipopolysaccharide recognition by the TLR4- MD-2 complex. Figure is reproduced from Park et al. Nature. (2009) 458, 1191-1195.
  • FIG. 71 illustrates the recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Figure is reproduced from Kang et al. Immunity. (2009) Dec 18;31 (6): 873-84.
  • FIG. 72 illustrates the crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide.
  • Figure is reproduced from Jin et al. Cell. (2007) Sep 21;130(6):1071-82.
  • FIG. 73 illustrates a comparison of Turbo adjuvant formulation with various TLR phenol extracted ViPS ligands.
  • FIG. 74 illustrates a comparison of Turbo adjuvant formulation with various TLR Typbar ligands.
  • FIG. 75 illustrates that Turbo as an adjuvant promotes antibody responses to haptenated protein antigens in all ages.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • An exemplary disease is a bacterial infection, and associated symptoms.
  • antibody means whole, intact antibody molecules, as well as fragments of antibody molecules that retain immunogen-binding ability, including the well- known active fragments F(ab')2, and Fab.
  • Antibodies are generated against antigens during the course of an immune response against a pathogen or immunogen and can include IgM and IgG, and all other IgG isotypes.
  • fragment is meant a portion of a polypeptide, polysaccharide, or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule, polysaccharide, or polypeptide.
  • binding to a molecule is meant having a physicochemical affinity for that molecule.
  • telomere binding partner present in a sample, but which ligand does not substantially recognize or bind other polypeptides in the sample.
  • decreases is meant a negative alteration of at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.
  • an effective amount of 1 is meant an amount of an immunogenic composition sufficient to induce or enhance an immune response in a subject.
  • Levels of induced immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by enzyme-linked immunosorbent assay, agglutination assay or any other method known in the art.
  • the effective amount of active compound(s) used to practice the present disclosure for prophylaxis or for therapeutic treatment of a disease varies depends upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living organism is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • immune response is meant the actions taken by a host to defend itself from pathogens or abnormalities.
  • the immune response includes innate (natural) immune responses and adaptive (acquired) immune responses.
  • Innate responses are antigen non-specific.
  • Adaptive immune responses are antigen specific.
  • An immune response in an organism provides protection to the organism against pathogenic infections when compared with an otherwise identical subject to which the composition or cells were not administered or to the human prior to such administration.
  • proliferation is used herein to refer to the reproduction or multiplication of similar forms, especially of bacterial or eukaryotic cells. That is, proliferation encompasses production of a greater number of bacterial or eukaryotic cells, and can be measured by, among other things, simply counting the numbers of bacterial or eukaryotic cells, measuring incorporation of 3 H- thymidine into bacterial or eukaryotic cells, and the like.
  • a "protective immune response" against an infectious disease refers to an immune response exhibited by a subject e.g., a mammal) that is protective against disease when the individual is subsequently exposed to and/or infected with wild-type bacteria or viruses.
  • the protective immune response results in detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing bacterial or viruses of the same strain and/or subgroup (and possibly also of a different, nonvaccine strain and/or subgroup) in vitro and in vivo.
  • creases is meant a positive alteration of at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.
  • infectious disease is meant a disease or condition in a subject caused by a pathogen that is capable of being transmitted or communicated to a non-infected subject.
  • infectious diseases include bacterial infections, viral infections, fungal infections, parasitic infections, and the like.
  • pathogen an infectious agent, such as a Salmonella enterica serovar Typhi, Salmonella enterica serovar Typhimurium (e. ., S. Typhi, S. Typhimurium), capable of causing infection, producing toxins, and/or causing disease in a host.
  • Salmonella enterica serovar Typhi Salmonella enterica serovar Typhimurium (e. ., S. Typhi, S. Typhimurium)
  • Salmonella enterica serovar Typhimurium e. ., S. Typhi, S. Typhimurium
  • sample refers to anything, which may contain the cells of interest (e.g, bacteria) for which a screening method or treatment is desired.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue.
  • biological tissue also include organs, tumors, lymph nodes, arteries, and individual cell(s).
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid, or the like.
  • a “subject” as used therein may be a human or non-human mammal.
  • Non-human mammals include, for example, primates, livestock, and pets, such as bovine, porcine, ovine, canine, feline, and murine mammals.
  • the subject is human.
  • reference is meant a standard or control condition.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison.
  • treat refers to reducing or improving an infectious disease or condition and/or one or more symptoms associated therewith. It will be appreciated that, although not precluded, treating an infectious disease or condition and/or one or more symptoms associated therewith does not require that the disorder, condition, disease, or symptoms associated therewith be completely ameliorated or eliminated.
  • vaccine is meant a composition, a lipid, a polysaccharide, a protein, or a nucleic acid of the disclosure, which serves to protect a subject against an infectious disease (e.g., bacterial disease) and/or to treat a subject having an infectious disease compared with an otherwise identical animal to which the vaccine is not administered or compared with the subject prior to the administration of the vaccine.
  • infectious disease e.g., bacterial disease
  • each recitation of the term “Turbo” independently refers to an exemplary non-limiting liposome adjuvant of the disclosure.
  • viralence is meant a degree of pathogenicity of a given pathogen or the ability of an organism to cause disease in another organism. Virulence refers to an ability to invade a host organism, cause disease, evade an immune response, and produce toxins.
  • virulent or “pathogenic” is meant a capability of a bacterium to cause a severe disease.
  • non-pathogenic is meant an inability to cause disease.
  • wildtype is meant a non-mutated version of a gene, allele, genotype, polypeptide, or phenotype, or a fragment of any of these. It may occur in nature or be produced recombinantly.
  • the term “derived from” encompasses, without limitation: an antigenic molecule that is isolated or obtained directly from an originating source (e.g. a bacterium, a virus, a fungus, a protozoan, or a multicellular parasite); a synthetic or recombinantly generated antigenic molecule that is identical or substantially related to an antigen from an originating source; or an antigenic molecule which is made from an antigen of an originating source or a fragment thereof.
  • an originating source e.g. a bacterium, a virus, a fungus, a protozoan, or a multicellular parasite
  • an antigenic molecule which is made from an antigen of an originating source or a fragment thereof e.g. a bacterium, a virus, a fungus, a protozoan, or a multicellular parasite
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the present disclosure provides immunogenic compositions useful for the induction of immune responses in subjects against antigens, particularly protein, polysaccharide, and polysaccharide-containing antigens.
  • the immunogenic compositions comprise liposomes comprised of an adjuvant (e.g., monophosphoryl Lipid A (MPLA), Di- or Tri-acyl lipopeptides (Pam2CSK4, and PamsCSK ⁇ , and combinations thereof), 1,2-dipalmitoyl-sn- glycero-3 phosphocholine (DPPC), and a protein, polysaccharide, or polysaccharide-containing antigen.
  • the compositions can further comprise a surfactant, such as but not limited to polyoxyethylene sorbitan monooleate.
  • compositions of the disclosure have unexpected efficacy in inducing antibody immune responses, particularly IgM and IgG based responses, against molecules (e.g., proteins and polysaccharides) which would be poorly immunogenic otherwise.
  • the disclosure provides immunogenic compositions useful for stimulating immune responses, particularly antibody responses against antigens including proteins, polysaccharides, or polysaccharide-containing antigens.
  • these compositions induce antibody responses in the absence of T cell help, and involve the stimulation of B cells, particularly Bl type B cells, which mediate long-lasting memory B cell responses.
  • B cells particularly Bl type B cells, which mediate long-lasting memory B cell responses.
  • Many bacterial and viral antigens make use of cell-surface glycosylation to aid in target cell infection and avoidance of host immunity.
  • targeting of an antibody response to cell surface polysaccharides and polysaccharide-protein conjugates enables the blocking of infection and immune elimination of pathogenic bacterial cells and viral particles prior to or early in an infection, especially prior to the onset of T cell immunity, which requires priming by antigen presenting cells and antigen-recognition (of infected cells, in the case of CD8+ T cell immunity).
  • an immunogenic composition comprising a liposome or nanosome adjuvant comprising an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof.
  • MPLA Monophosphoryl Lipid A
  • Di-acyl lipopeptide e.g. Pam2 CSK4
  • Tri-acyl lipopeptide e.g. Pam3 CSK4 or an analogue or derivative thereof, and any combination thereof.
  • MPLA is a derivative of lipid A, the naturally occurring hydrophobic group of the bacterial cell wall component lipopolysaccharide (LPS).
  • LPS covers the surface of most Gramnegative bacteria and is a highly immunogenic molecule, which is recognized by most mammalian immune systems by receptors of the toll-like receptor (TLR) family. In mammals, including humans, the TLR that recognizes LPS is TLR4. It is lipid A that is the immunogenic part of lipopolysaccharide.
  • MPLA is a synthetic structural analog of Lipid A, and is available as a number of derivates including, but not limited to, PHAD® and 3D(6-acyl) PHAD®.
  • the MPLA acts as an activator of immune cells by signaling through TLR4 and other receptors, and enhances the immunogenicity of the antigen by providing a so-called “danger signal” which activates immune cells to secrete inflammatory cytokines, chemokines, and other signaling molecules, all of which combine to enhance the development of an antibody response, including IgM, IgA, and IgG type antibodies against the antigen.
  • PanuCSh (Pam2CysSerLys4) is a synthetic diacylated lipopeptide (LP) and a potent activator of the pro-inflammatory transcription factor NF -KB.
  • Pan CSIGj mimics the acylated amino terminus of bacterial LPs and is recognized by the TLR2/TLR6 heterodimer.
  • the ParmCSI acts as an activator of immune cells by signaling through TLR2/6 and other receptors, thereby enhancing the immunogenicity of the antigen.
  • Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide (LP) and a TLR2/TLR1 ligand. It is a potent activator of the pro-inflammatory transcription factor NF-KB.
  • Pam3CSK.4 mimics the acylated amino terminus of bacterial LPs.
  • Bacterial LPs are a family of pro-inflammatory cell wall components found in both Gram-positive and Gram-negative bacteria. The stimulatory activity of these LPs resides in their acylated amino terminus. These bacterial LPs are recognized by TLR2, a receptor that plays a pivotal role in detecting a diverse range of pathogen-associated molecular patterns (PAMPs).
  • TLR2 forms a heterodimer with co-receptors TLR1 or TLR6, depending upon either tri- or diacylation of the ligand.
  • TLR2-TLR1 or TLR2-TLR6 a MyD88-dependent activation of NF-KB and AP-1 occurs, ultimately leading to an innate immune response.
  • Recognition of Pam 3 CSK4, a triacylated LP, is mediated by TLR2 which cooperates with TLR1 through their cytoplasmic domain to induce the signaling cascade leading to the activation of NF-KB.
  • any MPLA or Pam2CSK4 or Pam3CSK4 analogue or derivative that retains the ability to be recognized by TLR-family receptors is capable of being used in the immunogenic compositions of the disclosure, including but not limited to MPLA, PHAD®, 3D(6-acyl) PHAD®, 3D-MPLA, KDO DPLA, Pam 2 CSK 4 , or Pam 3 CSK 4 .
  • an immunogenic composition comprising a liposome or nanosome adjuvant comprising an effective amount of l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
  • DPPCs and their analogues or derivatives are amphipathic lipids, with hydrophilic heads, composed of the polar phosphatidylcholine group, and hydrophobic tails, formed by two nonpolar palmitic acid (Cl 6) chains.
  • DPPC or DPPC analogues/derivatives readily and spontaneously form micelles, monolayers, bilayers, liposomes, and nanosomes when in contact with polar solvents, such as water.
  • DPPC complexes with MPLA or MPLA-derivates or Pam 2 CSK4 or derivative, or Pam 3 CSK4 or derivatives to form larger structures encompassing MPLA and the antigens of the disclosure, including the proteins, polysaccharides, or polysaccharide-based molecules of the disclosure such that they are capable of enhancing the interaction with immune cells.
  • any analogue, derivative or variant of DPPC that is capable of forming micelles, monolayers, bilayers, liposomes, and nanosomes in polar solution is capable of being used in the compositions of the disclosure.
  • Examples of such analogues, derivatives, or variants include, but are not limited to l,2-distearoyl-sn-glycero-3- phosphocholine, l,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l -glycerol)], 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (succinyl), l,2-dimyristoyl-sn-glycero-3-phosphate, l,2-dimyristoyl-sn-glycero-3- phosphocholine, l,2-distearoyl-sn-glycero-3-phosphate, l,2-dipalmitoyl-sn-glycero-3 -phosphate, and l,2-dipalmitoyl-sn-glycero-3-
  • an immunogenic composition comprising composition comprising a surfactant such as polyoxyethylene sorbitan monooleate or an analogue or derivative thereof.
  • a surfactant such as polyoxyethylene sorbitan monooleate or an analogue or derivative thereof.
  • polysorbate 80 or tween 80 polyoxyethylene sorbitan monooleate is a nonionic surfactant and emulsifier that is commonly used in the art in a wide variety to pharmaceuticals, foods, and cosmetics.
  • polysorbate 80 or its analogues or derivatives aid in stabilizing the size of the liposomes or nanosomes which is optimal for interaction with immune cells.
  • Polysorbate 80 or its analogues or derivatives are also known to enhance the cellular ATP release at the site of injection, thus enhancing the immunogenicity of the compositions of the disclosure, and ultimately enhancing the antibody responses generated against the antigenic molecules of the disclosure. It is envisioned that in various embodiments of the disclosure, any polysorbate analogue or derivative molecule can be used in the compositions of the disclosure, including but not limited to polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 65, among others.
  • the present invention provides a compound or any salt, solvate, geometric isomer, or stereoisomer thereof.
  • the compound is disclosed in FIG. 6.
  • the compound is disclosed in Table 5, which discloses tripeptides, their corresponding aldehydes, and their corresponding oximes.
  • the compound is at least one of:
  • the compound is at least one of:
  • the compound is at least one of: Polysorbate 80 or a salt, solvate, geometric isomer, or stereoisomer thereof.
  • the compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R) or (S) configuration.
  • compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a compound illustrated herein by the racemic formula further represents either of the two enantiomers or mixtures thereof, or in the case where two or more chiral center are present, all diastereomers or mixtures thereof.
  • the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
  • salts embraces addition salts of free acids or bases that are useful within the methods of the disclosure.
  • pharmaceutically acceptable salt refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications.
  • the salts are pharmaceutically acceptable salts.
  • Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification, or formulation of compounds useful within the methods of the disclosure.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic, 2- hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic, P-hydroxybutyric, sal
  • Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium, and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N, N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • compositions and Formulations The disclosure provides pharmaceutical compositions comprising at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, which are useful to practice methods of the disclosure.
  • a pharmaceutical composition may consist of at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous or another route of administration.
  • a composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird.
  • Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically based formulations.
  • compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions.
  • a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.
  • a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • compositions suitable for ethical administration to humans are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at 15 least one compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e. ., RECOMB UMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable 20 carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • Formulations may be employed in admixtures with conventional excipients, i.e.. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of 5 administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or fragranceconferring substances and the like.
  • additional ingredients include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
  • the composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.
  • One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
  • the composition may include an antioxidant and a chelating agent which inhibit the degradation of the compound.
  • Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition.
  • Exemplary chelating agents include edetate salts (e.g., di sodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total 25 weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • antigens e.g., a protein or polysaccharide or polysaccharide- containing antigen
  • antigens combined with the immunogenic compositions described herein can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an immune response specific for the antigen (e.g., a polysaccharide or polysaccharide-containing antigen).
  • Also provided are methods of treating, ameliorating, and/or preventing a disease in a subject comprising immunizing the subject with a composition comprising an antigen (e.g., a protein, a polysaccharide, or polysaccharide-containing antigen) and the immuno-adjuvant of the disclosure.
  • a disease in a subject e.g., a bacterial disease
  • immunizing the subject with a composition comprising an antigen e.g., a protein, a polysaccharide, or polysaccharide-containing antigen
  • an antigen e.g., a protein, a polysaccharide, or polysaccharide-containing antigen
  • the methods include administering an immunologically effective amount of an antigen provided herein, and/or an immunologically effective amount of an immunogenic composition provided herein to an individual in a physiologically acceptable carrier.
  • the carrier or excipient for vaccines provided herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof.
  • a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
  • the methods also include administering an additional adjuvant, such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof, with the immunogenic composition of the disclosure.
  • an additional adjuvant such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof
  • a formulation for prophylactic administration also contains one or more adjuvants for enhancing the immune response to the antigens (e.g., polysaccharide or polysaccharide-containing antigens).
  • Suitable adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil, or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59.
  • compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, parenteral, pulmonary, intranasal, intravenous or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, and immunologically based formulations.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the cells into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • the cells of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical formulations of the cells of the disclosure include a therapeutically effective amount of the cells of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers which are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
  • compositions described herein can be introduced into a subject by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical formulation of the composition can be administered by inhalation or systemically, e.g., by intravenous injection.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the manifestation of symptoms associated with the disease or condition. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • Administration of the composition of the present disclosure to a subject may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or condition in the subject.
  • An effective amount of the composition necessary to achieve a therapeutic effect may vary according to factors such as the extent of implantation; the time of administration; the duration of administration; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder; age, sex, weight, condition, general health, and prior medical history of the subject being treated; and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • Routes of administration of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e. ., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • Suitable formulation of the composition sand dosages includes, for example, dispersions, suspensions, solutions, beads, pellets, magmas, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, aerosolized formulations for inhalation, compositions, and formulations for intravesical administration and the like.
  • kits for the treatment or prevention of a disease and/or disorder such as but not limited to a bacterial infection.
  • the kit includes a therapeutic or prophylactic composition containing an effective amount of an immunogenic composition (e.g., nanosomes or liposomes comprising Monophosphoryl Lipid A and 1,2- dipalmitoyl-sn-glycero-3 phosphocholine with a polyoxyethylene sorbitan monooleate surfactant) in unit dosage form.
  • an immunogenic composition e.g., nanosomes or liposomes comprising Monophosphoryl Lipid A and 1,2- dipalmitoyl-sn-glycero-3 phosphocholine with a polyoxyethylene sorbitan monooleate surfactant
  • the kit comprises a device (e.g., nebulizer, metered-dose inhaler) for immunogenic composition dispersal or a sterile container which contains a therapeutic or prophylactic immunogenic composition;
  • a device e.g., nebulizer, metered-dose inhaler
  • a sterile container which contains a therapeutic or prophylactic immunogenic composition
  • such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • an immunogenic composition of the disclosure is provided together with instructions for administering the immunogenic composition to a subject having or at risk of contracting or developing a bacterial or viral infection.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of a bacterial or viral infection.
  • the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of a bacterial or viral infection or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Characterization of an optimal B cell response.
  • Bib type B cells a subset of mature B cells which mediate long-lasting memory responses, especially the development of antigen-specific IgM and IgG responses, in the absence of T cellhelp (FIG. 1).
  • these studies found that Bib cells can expand concurrently with the resolution of bacteremia and persist for long time periods (FIG. 2) using Borrelia hermsii infection as a model.
  • Bib cells from convalescent mice but not from naive mice generated a specific antibody response and conferred long-lasting immunity, indicating that the protective immune response corresponds to Bib cell expansion and persistence as in the case of conventional B cell memory.
  • B cell responses to T cell-independent antigens are generated primarily by cross-linking B cell antigen receptors (BCR).
  • BCR B cell antigen receptors
  • Pneumococcal polysaccharide is also recognized by Bib cells, it does not induce antibody responses in X-linked immunodeficient mice (xid) mice, which have a mutation in the gene encoding for Bruton’s tyrosine kinase (Btk), which is required for optimal BCR-mediated signaling (illustrated in FIG. 3).
  • B. hermsii induces not only a specific antibody response but also a selective expansion of Bib cells in xid mice (FIG.2).
  • Example 2 Development of Turbo adjuvant for polysaccharide vaccines.
  • Bib cells generate antibody responses to a variety of bacterial antigens, including pneumococcal polysaccharide and the Vi polysaccharide (ViPS) of Salmonella Typhi, the causative agent of Typhoid in humans.
  • Many polysaccharide subunits vaccines lack adjuvants.
  • a few contain aluminum-based salts (Alum). None of the current ViPS subunit vaccines contain an adjuvant (FIG. 29). The recognition that stimulation of both TLR and BCR signaling could result in the generation of robust antibody responses against B.
  • hermsii antigens that was relatively long-lived in mouse models suggest the development of an immuno-adjuvant that can effectively present antigens such that optimal Bib responses are induced.
  • This adjuvant would be uniquely suited for plain polysaccharides and protein-conjugated polysaccharide vaccines, which would be present on a wide variety of bacterial and enveloped viral pathogens.
  • the potential to develop truly effective polysaccharide vaccines is especially relevant because they do not generate long- lasting immune responses despite multiple booster immunizations (FIGs. 7-8).
  • alum-based adjuvants which are well known in the art, induce a modest 2-fold increase in antibody responses whether or not the polysaccharide antigen is conjugated to a carrier protein (FIG. 9).
  • the data of the current disclosure show that the adjuvant formulation developed and disclosed herein (Turbo) induces a better response to ViPS conjugate vaccine compared to that induced by alum (FIG. 22).
  • Such an immune adjuvant would combine multiple immune signals to generate an optimal immediate antibody response that also provided long-lived immunity. These signals are illustrated in FIG. 28 and include initiation of the response via TLR4 signaling.
  • MPLA activates TLR4-MyD88 axis to induce pro IL-la and pro IL-10 (Signal 1).
  • the TLR4-Trif axis results in IRF3-mediated IFNy expression.
  • the IFNy signaling via IFNR is required for the induction Caspase 11.
  • MPLA alone does not induce activation of Caspase 1 or IL- 10 secretion and requires activation of inflammasome such as NLRP3.
  • inflammasome such as NLRP3.
  • MyD88 is also required for IL-1R1 signaling, it is possible that IL-la-IL-lRl-MyD88 signaling axis may be involved in B cells, in addition to MPLA-TLR4-MyD88 axis. These axes are expected to synergize with BCR signaling in antigen-specific B cells to upregulate B cell survival receptor BAFFR, plasma cell-differentiation receptor, TACI, and plOO, a precursor of NF-KB2.
  • NF-KB2 is central for CD40L-CD40-mediated expression of AID in B cells.
  • NF-KB2 is also required for BAFF-BAFFR-mediated survival of B cells through the expression of prosurvival molecules Mcl-1 & Bcl-XL.
  • One of the core components of the adjuvant is monophosphoryl lipid A (MPLA) (FIG. 13). While Lipid A is naturally the hydrophobic group of the bacterial cell wall component lipopolysaccharide, which covers the surface of most Gram-negative bacteria, it is lipid A that is the immunogenic part of lipopolysaccharide. MPLA is a synthetic structural analog of Lipid A, with one phosphate group instead of two (Lipid A has two phosphate groups) (FIG. 13) and is available commercially as PHAD® and 3D(6-acyl) PHAD®.
  • the MPLA of the current disclosure is combined with other molecules useful for specifically stimulating B cell responses, especially given that most T cells recognize peptide-based antigens as presented in the context of HLA or MHC complexes.
  • MPLA has several beneficial effects on adaptive immune responses, including antibody responses.
  • MPLA induces IgM, IgGl & IgG2c to Ova immunization as effectively as Alum or E6020 and also induces CD4 and CD8 responses comparable to that induced by LPS.
  • Shingrix (VZV), Fendrix (HepB) and Cervarix (HPV) vaccines which contain MPL® adjuvant in the forms of AS01 (3D6A MPLA from S.
  • MPLA offers several advantages for use in a clinical-grade immuno-adjuvant.
  • MPLA can be manufactured in a cGMP facility with high purity (>99%), improved stability and reduced manufacturing cost. Also, the lack of systemic toxicity and ability to manufacture it free of contamination by other bacterial components makes it amenable to a variety of adjuvant formulations.
  • MPLA was combined with l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC), an amphipathic lipid, with a hydrophilic head, composed of the polar phosphatidylcholine group, and hydrophobic tails, formed by two nonpolar palmitic acid (Cl 6) chains.
  • DPPC readily and spontaneously form micelles, monolayers, bilayers, and liposome when it is in contact with a polar solvent, such as water.
  • DPPC is also the main constituent of lung surfactants, reduces the surface tension of the alveolar liquid.
  • the final component is a surfactant which stabilizes the liposomes.
  • these studies used polyoxyethylene sorbitan monooleate or polysorbate 80. It was found that when used as a surfactant at a concentration above 0.5%, polysorbate 80 helps the stability of the size of the nanosomes/liposomes and enhances the cellular ATP release at the site of injection. Clinically, polysorbate 80 is already in use as an excipient in a majority of FDA- approved vaccines including for influenza (1.2 mg) (FIG. 14).
  • Example 3 In vivo use of Turbo to induce immune responses in mice.
  • Vi polysaccharide ViPS
  • ViPS conjugated to Tetanus toxoid a WHO preapproved vaccine against typhoid
  • Haptenated Ficoll a WHO preapproved vaccine against typhoid
  • Haptenated Ficoll a haptenated Chicken gamma globulins were used as the antigens (FIG. 15).
  • ViPS has a molecular formula of CioHieNOs and a molecular mass of 278.235.
  • ViPS consists of 600-10000 repeating units, and its O acetylation is about 95%. ViPS has been well characterized as a protective antigen and as a virulence factor. This is the principal immunogen in all typhoid subunit vaccines, which do not contain adjuvants. In fact, studies have indicated that contaminated LPS is likely to account for the immunogenicity of these vaccines (FIGs. 10, 11, 12, 49, 50 and 51).
  • FIG. 26 or 52 illustrates an example of one such mouse study wherein each animal received 3.4x107 nanosomes (containing 5 ug MPLA) with 0.02 - 2.5 ug ViPS in a total volume of 50 ul delivered intramuscularly.
  • a second booster dose was administered more than 28 days after the first immunization.
  • Peripheral blood was drawn at various timepoints in order to assess the concentration of specific antibody generated.
  • Turbo/ViPS would integrate various immune signals to induce activation of macrophages and dendritic cells (FIG. 28) as well as stimulating the activation of B cells by dendritic cells in B cell follicles or in the extrafollicular areas of the subject’s lymph nodes (FIG. 28).
  • mice received a number of doses ranging from 10 ug, 5 ug, 2.5 ug, and 1.25 ug of MPLA. Results showed observable but not significant correlations between antigen concentration of IgG down to 1.25 ug (FIG. 26A), the use of Turbo resulted in significant increases in IgG when used with even lower doses of antigen (FIG. 26B). These data suggested that Turbo was capable of inducing IgG responses even at sub-optimal antigen doses.
  • polysaccharide-based vaccines are of particular interest in pediatric patients, especially against meningococcal and pneumococcal diseases.
  • a series of mouse studies was conducted using single immunizations in relatively young (3 weeks) mice. The results from these studies showed that while IgM generation was comparably low in these animals regardless of Turbo use (FIG. 18A), the Turbo treated group demonstrated significantly higher concentrations of specific IgG up to 90 days post-vaccination (FIG. 18B).
  • animals were then challenged with . typhimurium strain RC60.
  • FIGs. 24A-24B illustrate conclusively that Turbodriven antibody responses are dependent on TLR4-MyD88 axis, as the ablation of either of these genes resulted in dramatically ameliorating the antibody response to vaccination.
  • mice were immunized with unconjugated ViPS vaccine (obtained from FDA) without and with Turbo and ViPS-specific antibody responses were measured. Results demonstrated that Turbo enhances antibody responses to both unconjugated (FIG. 54) and conjugated (ViPS-TT) vaccine (FIG. 55) in both inbred and outbred mice.
  • ViPS antigen is isolated from bacteria, and therefore, the ViPS preparations used in these studies is contaminated with bacterial components such as LPS.
  • ViPS from several sources was obtained and compared to LPS in mouse peritoneal macrophage activation studies.
  • the ViPS preparations compared were ViPS from Sanofi Pasteur, which was isolated from S. Typhi strain Ty2, ViPS from the US FDA, which was isolated from Citrobacter freundii strain WR7011, and ViPS from International Vaccine Institute, South Korea, which was isolated from S. Typhi, clinical isolate C6524.
  • FIG. 10 shows the results of these studies, which estimate that 2.5 ug of the ViPS dose given to mice as an immunogen is contaminated with ⁇ 2 ng of LPS.
  • mice deficient in TLR4 are impaired in responding to both plain ViPS vaccine (obtained from US FDA, lot 5) as well as WHO approved ViPS-TT conjugate vaccine (TypBar TCV from Bharat Biotech, India) (FIGs. 11 and 50B).
  • MyD88 knockout mice were found to exhibit a much lower response to another unconjugated ViPS vaccine (Typhim Vi), suggesting that this formulation contains contaminants that activate the TLR-related MyD88 pathway (FIG. 46).
  • FIGs. 12A and 12B demonstrate that while extracted ViPS is poorly immunogenic, the combination with Turbo can enhance immunogenicity with no difference in IgG levels between extracted and un-extracted Turbo groups. These data suggested that the presence of contaminating LPS in the polysaccharide does not appreciably contribute to the efficacy of Turbo as an immuno-adjuvant. As an additional proof of this observation, a similar study was conducted using the purely synthetic polysaccharide antigen NP-Ficoll.
  • NP-Ficoll is poorly immunogenic by itself, and does not induce a robust IgG2b or IgG2c response but can stimulate robust IgG responses when combined with Turbo (FIGs. 17A).
  • Example 4 Turbo induces robust immunity at all ages
  • FIGs. 30A-30C demonstrate the use of Turbo adjuvant in infant mice (10 days old) vaccinated with a ViPS tetanus toxoid conjugate antigen (ViPS-TT). Over the first 28 days, IgG levels in Turbo vaccinated mice were significantly higher than ViPS-TT antigen alone, and IgM levels were modestly increased (FIG. 30A). Animals were then followed out to 120 days, during which the significant increase in IgG levels in turbo-vaccinated mice continued (FIG. 30B). At the end of the experiments, mice were challenged with S. typhimurhmt strain RC60 bacteria and the persistence of live bacteria was assessed in blood, liver, and spleen. Turbo-vaccinated animals consistently demonstrated lower levels of bacteria (Fig 30C).
  • ViPS-TT ViPS tetanus toxoid conjugate antigen
  • FIG. 34 shows that Turbo vaccinated and boosted mice produced significantly more IgG as compared to mice receiving an alum-adj uvanted vaccine.
  • the beneficial effects of Turbo over alum were observed across multiple types of IgG, including IgGl, IgG2b, IgG2c, and IgG3 (FIGs. 35A-35B and 65A-65D).
  • Example 5 Turbo enhances two commercially available meningococcal vaccines
  • the ability of Turbo to enhance antibody responses to two commonly used meningococcal vaccines, Menveo® and MenQuadfi® was then assessed. Mice were immunized with two approved meningococcal polysaccharide vaccines (i.e., Menveo® from GSK and MenQuadfi® from Sanofi Pasteur). These vaccines are multivalent, and contain four serotype polysaccharides, namely A, C, W and Y. The results of these studies demonstrated that Turbo enhances both IgM (FIGs. 43 and 61) and IgG (FIGs. 44, and 61) antibody responses to both vaccines and all four serotypes with a single immunization.
  • FIGs. 37A and 37B As an initial study, Turbo was combined with a number of TLR4 agonists to determine which could combine with Turbo to promote greater antibody production to both tetanus toxoid conjugated and unconjugated ViPS antigen.
  • FIGs. 37A and 37B As an initial study, Turbo was combined with a number of TLR4 agonists to determine which could combine with Turbo to promote greater antibody production to both tetanus toxoid conjugated and unconjugated ViPS antigen.
  • chlodronate liposomes were used to deplete macrophages before Turbo ViPS/ViPS-TT vaccination.
  • depletion of macrophages significantly decreased IgG production in treated animals (FIG 4 IB).
  • FIGs. 46 and 50 shows that Turbo enhances antibody response to the unconjugated ViPS vaccine Typhim Vi.
  • FIG. 47 demonstrates that Turbo enhances antibody response to unconjugated ViPS vaccine in both inbred and outbred mice and FIG. 48 illustrates that Turbo likewise enhances antibody response to conjugated ViPS vaccine (ViPS-TT) in both inbred and outbred mice.
  • TLR4 Toll-Like Receptor 4
  • HEK293 cells expressing a specific TLR or NOD-like receptor (NLR) with an NF-KB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were incubated with Typhim Vi® (5 pg ViPS/ml l/5th of human dose of lot V2A451M from Sanofi Pasteur), Typbar TCV® (5 pg ViPS/ml l/5th of human dose of lot 76B21035A from Bharat Biotech), and Vi PS (33 pg ViPS/ml lot 5 PDML158299 from US FDA).
  • Typhim Vi® 5 pg ViPS/ml l/5th of human dose of lot V2A451M from Sanofi Pasteur
  • Typbar TCV® 5 pg ViPS/ml l/5th of human dose of lot 76B21035A from Bharat Biotech
  • Vi PS 33 pg ViPS/ml lot 5 PDML158299 from US FDA.
  • Positive controls were 10 8 heat-killed Listeria monocytogenes cells/ml for m/hTLR2, Poly(I:C) HMW at 1 pg/m for m/hTLR3, E. coli K12 LPS at 100 ng/ml for m/hTLR4, S.
  • typhimurium flagellin at 100 ng/ml m/hTLR5, CL307 at 1 pg/ml m/hTLR7: TL8-506 at 1 pg/ml for hTLR8, TL8-506 at 10 pg/ml for mTLR8, CpG ODN 2006 at 10 pg/ml for hTLR9, CpG ODN 1826 at 1 pg/ml for mTLR9, C12-iE-DAP at 10 pg/ml for m/hNODl, and L18-MDP at 1 pg/ml m/hNODl.
  • HEK -Blue Null cell lines which do not express any TLR or NLR
  • the media containing HEK-Blue DetectionTM is designed for the detection of NF-KB induced SEAP expression. After a 16-24-hour incubation the optical density (OD) is read at 650 nm. Results demonstrated that all three vaccine types strongly stimulated human and mouse TLR4, and ViPS also demonstrated significant stimulation of mouse TLR2.
  • FIG. 5 IB Peritoneal exudate cells were incubated with indicated stimulants for 24 h and IL-6 levels in the supernatant was measured by ELISA.
  • FIG. 51C Wildtype mice were immunized i.m.
  • ViPS-specific IgM and IgG levels were measured by ELISA. Results demonstrated a significant loss of immunogenicity when TLR4 signaling is abrogated.
  • Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
  • Vi Polysaccharide vaccine (FIG. 54).
  • male and female mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA).
  • Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
  • mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of Typbar TCV® vaccines containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA).
  • Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
  • mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of either Typhim Vi vaccine or Typbar TCV vaccine containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA).
  • Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA. Both studies found robust Turbo efficacy in both male and female mice.
  • TLR4 and MyD88 signaling were then examined (FIG. 59).
  • Wildtype (C57BL/6J) male and female mice of TLR4-/- and MyD88-/- genotype were immunized i.m with 50 pl of Menveo® (4 micrograms of MenACWY) or MenQuadfi® (2.5 micrograms of MenACWY) or with and Turbo (containing 5 pg of MPLA).
  • Meningococcal serotype-specific IgM and IgG levels were then measured by ELISA and the results demonstrated that immunogenicity is dependent largely on the function of MyD88, rather than TLR4 specifically.
  • MyD88 is an adaptor protein that functions in the signaling through multiple TLR family receptors.
  • One significant potential clinical use of Turbo adjuvanted vaccines would be in the vaccination of the very young.
  • wildtype (C57BL/6J) male and female mice 9 days of age (infant) were immunized subcutaneously with 25 microliters of Menveo® (2 micrograms of MenACWY) or MenQuadfi® (1.25 micrograms of MenACWY) or with and without Turbo (containing 2.5 pg of MPLA) (FIG. 60).
  • Meningococcal serotype-specific IgM and IgG levels were then measured by ELISA.
  • Wildtype (C57BL/6J) male and female mice were immunized i.m with 50 pl of phenol- extracted ViPS (2.5 micrograms) admixed with Turbo (containing 5 pg of MPLA, known TLR4 agonist); Turbo (containing 5 pg of Pam2CSK4, a known TLR2/6 agaonist); or Turbo (containing 5 pg of Pam CSK , a known TLR1/2 agaonist); ViPS-specific IgM and IgG isotype levels were then measured by ELISA.
  • Turbo containing 5 pg of MPLA, known TLR4 agonist
  • Turbo containing 5 pg of Pam2CSK4, a known TLR2/6 agaonist
  • Turbo containing 5 pg of Pam CSK , a known TLR1/2 agaonist
  • ViPS-specific IgM and IgG isotype levels were then measured by ELISA.
  • the TLR4 ligand MPLA generated the widest variety of antibody generation by stimulating significantly more IgM, IgG, IgG2b, IgG2c, and IgG3 than the other agonists, however PamsCSIGi induced a greater amount of IgGl, an equivalent amount of IgG and IgG2c to MPLA.
  • Pai CSIGi generated an equivalent amount of IgG3 to MPLA but was much lower than MPLA in every other immunoglobulin type tested.
  • the studies in FIG. 74 sought to compare PaimCSK-t to Pan CSB using the Typbar TCV ViPS and tetanus toxoid conjugate vaccine. Both formulations generated roughly similar levels of immunoglobulin that were not significantly different except IgG3, which Pam2CSK4 generated greater levels of.
  • RVGP Rabies virus glycoprotein
  • Fig. 66 Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the right thigh of the hind limb with the model antigen NP-CGG, or protein conjugated- Vi PS vaccine (Typbar TCV) that were bedside mixed without or with Turbo. In a group of mice where the antigen was administered in the right thigh, and the adjuvant was injected in the left thigh (i.e., contralateral). NP-specific antibody and ViPS-specific antibody levels were then measured by ELISA. Results demonstrated that the adjuvant properties of Turbo require ipsilateral administration with the vaccine antigen.
  • Embodiment 1 provides an immunogenic composition.
  • the composition comprises one or more antigenic molecules between 0.01 mg/ml and 50.0 mg/ml.
  • the composition comprises a surfactant at a concentration of at least 0.5% (w/w).
  • the composition comprises an effective amount of a liposome adjuvant.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam 2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pams CSK4) or an analogue or derivative thereof, and any combination thereof, and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
  • MPLA Monophosphoryl Lipid A
  • Di-acyl lipopeptide e.
  • Embodiment 2 provides the immunogenic composition of embodiment 1, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • Embodiment 3 provides the immunogenic composition of embodiment 2, wherein the polysorbate 80 or an analogue or derivative thereof is between 0.1% and 10% (w/w).
  • Embodiment 4 provides the immunogenic composition of any one of embodiments 1-3, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a protein, a peptide, a peptide/polysaccharide conjugate, a protein/polysaccharide conjugate and any combination thereof.
  • Embodiment 5 provides the immunogenic composition of any one of embodiments 1-4, wherein the antigenic molecule is derived from a bacterium.
  • Embodiment 6 provides the immunogenic composition of any one of embodiments 1-4, wherein the antigen molecule is derived from a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite.
  • a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite.
  • Embodiment 7 provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition.
  • the composition comprises one or more antigenic molecules.
  • the composition comprises a surfactant at a concentration of at least 0.5% (w/w).
  • the composition comprises an effective amount of a liposome adjuvant.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g.
  • Pair CSK4 or an analogue or derivative thereof
  • Tri-acyl lipopeptide e.g. Paim CSK4 or an analogue or derivative thereof, and any combination thereof
  • DPPC l,2-dipalmitoyl-sn-glycero-3 phosphocholine
  • Embodiment 8 provides the method of embodiment 7, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
  • Embodiment 9 provides the immunogenic composition of embodiment 8, wherein the polysorbate 80 is between 0.1% and 10% (w/w).
  • Embodiment 10 provides the method of any one of embodiments 7-9, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a peptide, and a peptide/polysaccharide conjugate.
  • Embodiment 11 provides the method of any one of embodiments 7-10, wherein the antigenic molecule is derived from a bacterium.
  • Embodiment 12 provides the method of any one of embodiments 7-11, wherein the antigenic molecule is derived from a virus.
  • Embodiment 13 provides the method of any one of embodiments 7-12, wherein the immune response is independent of T cells.
  • Embodiment 14 provides the method of any one of embodiments 7-12, wherein the immune response is dependent on T cells.
  • Embodiment 15 provides the method of any one of embodiments 7-14, wherein the immune response is B cell mediated.
  • Embodiment 16 provides the method of embodiment 15, wherein the B cells are stimulated by B cell receptor and toll-like receptor (TLR) signaling.
  • TLR toll-like receptor
  • Embodiment 17 provides the method of any one of embodiments 15-16, wherein the B cells are Bib cells or Bib equivalent cells.
  • Embodiment 18 provides the method of any one of embodiments 7-16, wherein the immune response comprises the generation of antigen-specific antibodies.
  • Embodiment 19 provides the method of embodiment 18, wherein the antibodies comprise IgM antibodies, IgG antibodies, or a combination of IgM and IgG antibodies.
  • Embodiment 20 provides the method of any one of embodiments 7-18, wherein the immune response generates long-term immune memory.
  • Embodiment 21 provides the method of any one of embodiments 7-20, wherein the subject is human, primate, bovine, porcine, ovine, canine, feline or murine.
  • Embodiment 22 provides a kit comprising the immunogenic composition of any one of embodiments 1-6.
  • Embodiment 23 provides a method of treating, ameliorating, and/or preventing a disease in subject in need thereof, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of embodiment 1-6.
  • Embodiment 24 provides the method of embodiment 23, wherein the disease is associated with the antigenic molecule.
  • Embodiment 25 provides the method of any one of embodiments 23-24, wherein the disease is a polysaccharide encapsulated bacterial infection.
  • Embodiment 26 provides the method of embodiment 25, wherein the polysaccharide encapsulated bacterial infection is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiritidis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptococcus, Pseudomonas aeruginosa, Klebsiella, Pasteurella, Brucella, Francisella, Helicobacter, Vibrio and Bacillus anthracis.
  • the polysaccharide encapsulated bacterial infection is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiritidis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptoc
  • Embodiment 27 provides the method of any one of embodiments 23-26, wherein the bacterial infection is a non-polysaccharide encapsulated bacterial infection.
  • Embodiment 28 provides the method of embodiment 27, wherein the non-polysaccharide encapsulated bacterial infection is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Streptococcus, Chamydia, Escherichia coli, Meningococcal group B, Staphylococcus aureus, and Group A and B Streptococcus.
  • the non-polysaccharide encapsulated bacterial infection is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Strepto

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Abstract

The present disclosure provides immunogenic compositions useful for inducing immunity to polysaccharide antigens and/or and polysaccharide-containing antigens. Also included are methods of stimulating an immune response in subjects in need thereof and methods of treating, ameliorating, and/or preventing diseases and/or disorders in subjects comprising administering the immunogenic compositions of the disclosure.

Description

IMMUNE ADJUVANTS FOR POLYSACCHARIDE VACCINES
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/431,247, fded December 8, 2022, and U.S. Provisional Patent Application No. 63/469,951, filed May 31, 2023, each of which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under All 59798 awarded by National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
Vaccination is a cornerstone strategy for inducing broad protective immunity against a range of viral and bacterial pathogens in both animal and human subjects. While vaccines focusing on protein targets are well established, vaccines targeting polysaccharide or polysaccharide/protein conjugates have also been the subject of research interest. One reason for the attractiveness of this strategy is the fact that many infectious agents, particularly bacteria and some enveloped viruses, express surface polysaccharides that play key roles in host immune evasion and pathogenesis. As such, directing host antibody immune responses against these targets would act to block target cell infection and mark bacterial cells and viral particles for subsequent immune system -mediated clearance. Despite the promise of this immune system strategy, polysaccharides by themselves are poorly immunogenic. Even, even when combined with conjugated proteins and/or traditional, human-compatible adjuvants, polysaccharides sometimes fail to generate long-term antibody and T cell responses, despite repeated booster immunizations.
As such, a need exists in the art for immune compositions and methods which increase the immunogenicity of antigens including polysaccharides and proteins and are capable of stimulating effective, long-lived antibody and T cell responses in both human and animal subjects. The present disclosure addresses these unmet needs. SUMMARY OF THE INVENTION
As described herein, the present disclosure relates to immunogenic compositions useful for inducing immunity to polysaccharide antigens and/or and polysaccharide-containing antigens. Also included are methods of stimulating an immune response in subjects in need thereof and methods of treating, ameliorating, and/or preventing diseases and/or disorders in subjects comprising administering the immunogenic compositions of the disclosure.
In one aspect, the present invention provides an immunogenic composition comprising: one or more antigenic molecules between 0.01 pg/ml and 50.0 pg/ml; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or a derivative thereof.
In certain embodiments, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
In certain embodiments, the polysorbate 80 or an analogue or derivative thereof is between 0.1% and 10% (w/w).
In certain embodiments, the antigenic molecule is selected from the group consisting of a polysaccharide, a protein, a peptide, a peptide/poly saccharide conjugate, a protein/polysaccharide conjugate and any combination thereof.
In certain embodiments, the antigenic molecule is derived from a bacterium.
In certain embodiments, the antigenic molecule is derived from a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite.
In another aspect, the present invention provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more antigenic molecules; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
In certain embodiments, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
In certain embodiments, the polysorbate 80 is between 0.1% and 10% (w/w).
In certain embodiments, the antigenic molecule is selected from the group consisting of a polysaccharide, a peptide, and a peptide/polysaccharide conjugate.
In certain embodiments, the antigenic molecule is derived from a bacterium.
In certain embodiments, the antigenic molecule is derived from a virus. In certain embodiments, the immune response is independent of T cells. In certain embodiments, the immune response is dependent on T cells. In certain embodiments, the immune response is mediated by B cells.
In certain embodiments, B cells are stimulated by B cell receptor and toll-like receptor (TLR) signaling.
In certain embodiments, the B cells are Bib cells or Bib equivalent cells.
In certain embodiments, the immune response comprises the generation of antigenspecific antibodies.
In certain embodiments, the antibodies comprise IgM antibodies, IgG antibodies, or a combination of IgM and IgG antibodies.
In certain embodiments, the immune response generates long-term immune memory.
In certain embodiments, the subject is human, primate, bovine, porcine, ovine, canine, feline or murine.
In another aspect, the current disclosure provides a kit comprising the immunogenic composition of any one of claims 1-6.
In another aspect, the current disclosure provides a method of treating, ameliorating, and/or preventing a disease in subject in need thereof, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein.
In certain embodiments, the disease is associated with the antigenic molecule.
In certain embodiments, the disease is a polysaccharide encapsulated bacterial infection.
In certain embodiments, the polysaccharide antigen is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiriti dis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptococcus, Pseudomonas aeruginosa, Klebsiella, Pasteurella, Brucella, Francisella, Helicobacter, Vibrio and Bacillus anthracis.
In certain embodiments, the bacterial infection is a non-polysaccharide encapsulated bacterial infection.
In certain embodiments, the non-polysaccharide antigen is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Streptococcus, Chamydia, Escherichia coli, Meningococcal group B, Staphylococcus aureus, and Group A and B Streptococcus.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, there are shown in the drawings exemplary embodiments. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
FIG. l is a table illustrating the various B cell subsets in mice.
FIG. 2 illustrates that a long term Bib cell expansion is concurrent with the resolution of bacteremia in mice deficient in Btk, a kinase required for BCR-mediated signal: Involvement of a co-stimulatory signal.
FIG. 3 is a diagram illustrating B cell receptor (BCR) signaling, highlighting the role of Btk, a kinase for signal transduction.
FIG. 4 is a diagram illustrating Toll-Like Receptor (TLR) signaling highlighting the role of MyD88, an adaptor for signal transduction of multiple members of the TLR family. FIG. 5 is a diagram illustrating the critical role for the PH domain of Btk for docking to the membrane and kinase domains of Btk for the autophosphorylation, that is essential for Btk’s function.
FIGs. 6A-6B illustrate that MyD88 and Btk mediated signaling are essential for T cellindependent pathogen-specific IgM immune responses. One of the mouse strains has a substitution mutation in PH domain (i.e., R28C) and the other one has a mutation/deletion in the kinase domain of the Btk protein. FIG. 6A illustrates the levels of IgM after stimulation in indicated mouse lines. FIG. 6B illustrates bacterial cells present in peripheral blood in wildtype or double knockout (DKO, deficient in both Btk and Myd88) mice.
FIGs. 7A-7B illustrate that plain polysaccharide vaccines induce poor and short-lived antibody response in adults. FIG. 7A is a table comparing seropositivity rates (%) and geometric mean titers (GMTs) of antigen-specific antibody during the first 12 months after vaccination with two indicated polysaccharide vaccines. FIG. 7B displays similar data in graph form.
FIGs. 8A-8H illustrate that polysaccharide conjugated to a widely used carrier proteins such as CRM197 for the production of conjugate vaccines does not induce efficient response in Typhoid and invasive pneumococcal disease endemic countries. Graphs show GMC, as measured by ELISA in adults in Pakistan (FIG. 8A), India (FIG. 8B), children in Pakistan (FIG. 8C) and India (FIG. 8D), older infants in Pakistan (FIG. 8E) and India (FIG. 8F), and infants in Pakistan (FIG. 8G) and India (FIG.8H).
FIG. 9 is a table illustrating that Alum-based adjuvants induce a modest (~2-fold) increase in antibody response to Vi PS conjugate vaccines.
FIG. 10 is a diagram illustrating that the ViPS antigen is isolated from bacteria. Therefore, the ViPS preparation is contaminated with bacterial components such as LPS. ViPS from Sanofi Pasteur was isolated from S. Typhi strain Ty2, ViPS from US FDA was isolated from Citrobacter freundii strain WR7011. ViPS from International Vaccine Institute, South Korea was isolated from S. Typhi, clinical isolate C6524. Mouse peritoneal macrophages were incubated with various concentrations of ViPS for three sources or LPS (from Sigma Aldrich as a positive control). The levels of IL-6 as a readout were measured by ELISA.
FIGs. 11A-11C show that mice deficient in TLR4, which recognizes LPS are impaired in responding to plain ViPS vaccine (obtained from US FDA, lot 5) as well as World Health Organization (WHO) pre-approved ViPS-Tetanus Toxoid conjugate vaccine (ViPS-TT, commercially known as TypBar TCV from Bharat Biotech, India). This suggests that LPS present in these vaccines plays a significant role in antibody responses.
FIGs. 12A-12B illustrate that Phenol extracted ViPS is poorly immunogenic. However, its immunogenicity can be promoted by Turbo, which refers to a non-limiting liposome adjuvant of the disclosure. ViPS (obtained from US FDA) was subjected to phenol extraction protocol to eliminate the TLR ligand. C57BL6 mice were immunized i.m. with original stock ViPS (2.5 pg) or phenol-extracted ViPS (2.5 pg) with and without Turbo (5 pg), and levels of ViPS-specific (FIG. 12 A) IgM and (FIG. 12B) IgG were measured by ELISA.
FIG. 13 are diagrams comparing the structures of bacterially derived Lipid A (top) and two synthetic Lipid A molecules (bottom).
FIGs. 14A-14C are diagrams of the components of the immune adjuvant of the current disclosure. FIG. 14A illustrates monophosphroyl Lipid A (MPLA), l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC), and polyoxyethylene sorbitan monooleate (Polysorbate 80). FIG. 14B is a diagram of an example liposome comprised of the composition of the disclosure. FIG. 14C is a chart of liposome size as compared to concentration of particles. D IO: The portion of particles with diameters smaller than this value is 10%. D50: The portions of particles with diameters smaller and larger than this value are 50%. Also known as the median diameter. D90: The portion of particles with diameters below this value is 90%.
FIG. 15 is a diagram of a typical immunization strategy for a mouse study using the Turbo adjuvant of the disclosure with various immunogens.
FIGs. 16A-16B: FIG. 16A illustrate that Turbo driven IgM and IgG isotype responses are independent of mouse background. C57BL6, 129Sv or BALB/c mice were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo (5 pg) and levels of ViPS-specific IgM and IgG were measured by ELISA. FIG. 16B illustrate that Turbo also enhances IgM and IgG isotype responses to ViPS-TT conjugate vaccine.
FIGs. 17A-17B: FIG. 17A illustrate that Turbo also enhances IgM and IgG isotype responses to 4-Hydroxy-3 -nitrophenylacetyl (NP) hapten conjugated to Ficoll (NP-Ficoll), a high molecular weight synthetic polysaccharide widely used for studying T cell-independent B cell activation. FIG. 17B illustrate that Turbo also enhances IgM and IgG isotype responses to NP conjugated to Chicken Gamma Globulin (NP-CGG), a model antigen for studying T cell- dependent B cell responses. Unlike Alum which activate NLPR3 inflammasome, and polarizes antibody response towards IgGl, Turbo promoted IgGl, IgG2b, IgG2c/IgG2a, and IgG3, suggesting wide range of Fc receptor- and complement-mediated protective mechanisms are possible with Turbo as an adjuvant.
FIGs. 18A-18B illustrate that a single immunization of ViPS with Turbo induces long- lasting and sustained IgG response increases protective immunity in young (3 -week-old) mice. C57BL6 mice (3-week-old) were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo (5 pg) and levels of ViPS-specific IgM and IgG were measured by ELISA. Statistical differences were determined using Two-way ANOVA with Bonferroni post-test. ** denotes p<0.01. (FIG 18B). On day 90, mice were infected i.p. with 3 x 104 CFUs of ViPS expressing S. Typhimurium strain RC60 and three days post-challenge bacterial burden in the blood was determined. Each dot represents an individual mouse, and the bar represents median. Statistical differences were determined by Mann-Whitney test. **** p<0.001; *p<0.05 and N.S. denotes not significant.
FIG. 19 illustrate that Turbo promotes IgG response in infant mice. Ten-day old C57BL6 mice were immunized i.m. with ViPS-TT conjugate vaccine (1.0 pg) alone, admixed with Turbo (2.5 pg), and levels of ViPS-specific IgG was measured by ELISA.
FIGs. 20A-20B illustrate the use of Turbo vaccination in mice with and without a squalene-based adjuvant (SE). C57BL6 mice (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) alone, admixed with Turbo (5 pg), 2% Squalene-emulsion (SE) or both, and levels of ViPS-specific IgM (FIG. 20A) and IgG (FIG. 20B) were measured by ELISA. Statistical differences were determined using Two-way ANOVA. **** p<0.001, **p<0.01 and N.S. denotes not significant.
FIG 21 illustrates the comparison of various Turbo formulations. The structures of all the three TLR4 agonists are shown in Figure 13. C57BL/6 mice (8-12-week-old) were immunized i.m. with ViPS vaccine (2.0 pg) alone, admixed with Turbo (5 pg), and levels of ViPS-specific IgM and IgG isotypes were measured by ELISA.
FIGs. 22A-22B illustrate the comparison of various Turbo formulations and other adjuvants namely alum and squalene-based adjuvant (SE). C57BL/6 mice (8-12-week-old) were immunized i.m. with ViPS-TT conjugate vaccine (2.0 pg) alone, admixed with Turbo (5 pg), 2% Squalene-emulsion (SE) or Alum at 0.5 mg (0.25 mg aluminum hydroxide & 0.25 mg magnesium hydroxide), and levels of ViPS-specific IgM (FTG. 22A) and IgG (FIG. 22B) were measured by ELISA.
FIGs. 23A-23B illustrate that Turbo-driven antibody responses are not dependent on NLRP3 inflammasome or pyropotosis. Wildtype or mice deficient in NLPR3, Caspl, or GsdmD on C57BL6 background (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) admixed with Turbo (5 pg), and levels of ViPS-specific IgM (FIG. 23 A) and IgG (FIG. 23B) were measured by ELISA.
FIGs. 24A-24B illustrate that Turbo-driven antibody responses is dependent on TLR4- MyD88 axis. Wildtype or mice deficient in TLR4, or TLR adaptor proteins MyD88 or Trif on C57BL6 background (8-12-week-old) were immunized i.m. with ViPS (2.5 pg) admixed with Turbo (5 pg), and levels of ViPS-specific IgM (FIG. 24A) and IgG (FIG. 24B) were measured by ELISA. Statistical differences were determined using Two-way ANOVA. **** p<0.001, **p<0.01.
FIGs. 25A-25B illustrate that Turbo induces increased primary and secondary anti-ViPS IgG response, suggesting that turbo promotes IgM and IgG memory B cells. Mice were reimmunized 62 days after primary immunization to measure the booster response.
FIGs. 26A-26B illustrate the induction of anti-ViPS IgG response under antigen-limiting conditions by Turbo. C57BL6 mice were immunized i.m. with ViPS (2.5 pg) or ViPS (2.5 pg) + Turbo at various concentrations (FIG. 26A); or with ViPS at various concentrations + Turbo at 5 pg (FIG. 26B). ViPS-specific IgG was measured by ELISA.
FIGs. 27A-27B illustrate that Turbo vl & v2 (without and with 2% Tween 80, respectively) induces increased anti-ViPS IgG response.
FIG. 28 is a schematic of a proposed model illustrating the integration of various molecular signals and cellular interactions.
FIG. 29 is a table illustrating that many approved polysaccharide subunit vaccines lack adjuvants.
FIGs. 30A-30C are graphs that illustrate that Turbo, when used as an adjuvant, promotes a rapid and robust antibody response in infant (10 days old) mice. Here the vaccine comprised ViPS-TT (TypBar TCV) conjugate as an antigen. FIG. 30A illustrates the IgM (top) and IgG (bottom) levels in each individual animal at the indicated time post-immunization. FIG. 30B illustrates the same data in graphical form and at timepoints out to 120 days. FIG. 30C illustrates the levels of RC60 in blood, liver, and spleen of vaccinated animal after challenge.
FIG. 31 are graphs that illustrate that Turbo promotes a sustained antibody response in adult (2 - 4 months old) mice when vaccinated with an unconjugated ViPS vaccine. Graphs indicate IgM (left) and IgG (right) levels in peripheral blood at the indicated timepoints.
FIG. 32 are graphs that illustrate that Turbo adjuvant promotes all IgG isotypes in adult mice vaccinated with a ViPS-TT (TypBar TCV) conjugate vaccine. Two mouse strains were used, C57BL/6J (top) and 129Sl/SvImJ (bottom).
FIG. 33 are graphs that illustrate that Turbo adjuvant promotes a rapid and robust antibody response in aged mice (1.6 years old) to ViPS-TT (TypBar TCV) conjugate vaccine.
FIG. 34 are graphs that illustrate that Turbo promotes heightened recall responses to booster immunization, indicating the generation of B cell memory.
FIGs. 35A-35B are graphs that illustrate that Turbo adjuvant promotes long-lasting antibody responses, which indicates the generation of antigen-specific, long-lived plasma cells. Graphs indicate the comparison of Turbo (filled black dots) with alum (open red dots).
FIG. 36 are graphs that illustrate that Turbo promotes a rapid and robust antibody response in infant (10 days old) mice challenged with hapten-conjugated antigens.
FIGs. 37A-37B are graphs that illustrate that a wide range of TLR4 agonists combine with Turbo adjuvant to generate significant immunity to both conjugated and unconjugated ViPS vaccines. FIG. 37A depicts unconjugated ViPS antigen. FIG. 37B depicts a ViPS-tetanus toxoid conjugate vaccine.
FIGs. 38A-38B are graphs that show that Turbo adjuvant function is independent of NLRP3 inflammasome or pyroptosis mechanisms. FIG. 38A illustrates data in various knockout mice using unconjugated ViPS as the immunogen. FIG. 38B illustrates data in the same mice using phenol extracted ViPS antigen.
FIG. 39 are graphs that illustrate that Turbo adjuvant engages TLR4-MyD88, TLR4-Trif, and Caspase 11 signaling axes when administered with a ViPS-TT conjugate vaccine.
FIGs. 40A-40B are graphs that illustrate that conventional dendritic cells (eDCs) are not required for Turbo adjuvant function. FIG. 40A depicts the use of an unconjugated ViPS antigen. FIG. 40B depicts use of a conjugated ViPS-tetanus toxoid vaccine. FIGs. 41 A-41B are graphs that illustrate that macrophages but not neutrophils are required for the adjuvant effects of Turbo. FIG. 41A illustrates use of an unconjugated ViPS antigen. FIG. 42B illustrates use of a tetanus toxoid conjugated vaccine.
FIG. 42 is a schematic of a macrophage-centric model for Turbo adjuvant function.
FIG. 43 are graphs that illustrate that Turbo enhances serotype-specific IgM responses to meningococcal polysaccharide conjugate vaccines Menveo and MenQuadfi.
FIG. 44 are graphs that illustrate that Turbo enhances serotype-specific IgG responses to meningococcal polysaccharide conjugate vaccines Menveo and MenQuadfi.
FIG. 45 are graphs that illustrate that Turbo adjuvanticity to ViPS conjugate vaccine is Trif pathway dependent, and admixing Turbo to the vaccine is required for the optimal antibody response.
FIG. 46 are graphs that illustrate that Turbo enhances antibody response to unconjugated ViPS vaccine (Typhim Vi) in a TLR4 and Myd88 dependent manner.
FIG. 47 are graphs that illustrate that Turbo enhances antibody response to unconjugated ViPS vaccine in both inbred and outbred mice.
FIG. 48 are graphs that illustrate that Turbo enhances antibody response to conjugated ViPS vaccine (ViPS-TT) in both inbred and outbred mice.
FIG. 49 illustrates that typhoid Vi Polysaccharide subunit vaccines Typhim Vi® and Typbar TCV® stimulate mouse and human Toll-Like Receptor 4 (TLR4). Statistics done using unpaired t test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001.
FIGs. 50A-50B illustrate that the immunogenicity of Typhim Vi® and Typbar TCV® is dependent on TLR4 and TLR4 signaling adaptors MyD88 and Trif. Wildtype (C57BL/6I), TLR4-/-, MyD88-/-, or Trif-/- (n = 6-7) on C57BL6 background male and female mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of (FIG. 50A) Typhim Vi® or (FIG. 50B) Typbar TCV® vaccines containing 2.5 pg of Vi PS. ViPS-specific IgM, IgGl, IgG2b, IgG2c, and IgG3 levels were measured by ELISA. Statistics done using 2- way ANOVA Sidak’s multiple comparisons test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIGs. 51A-51C illustrate the elimination of immunostimulatory components in the ViPS preparation results in the loss of immunogenicity. FIG. 50A. Wildtype (C57BL/6J) or TLR4-Z- mice 8-10-week-old of both sexes (n=6) were immunized i.m. with 50 pl 2.5 ig of ViPS (lot 5 PDML 158299 from US FDA), and ViPS-specific IgM and IgG levels were measured by ELISA. (FIG. 50B) Peritoneal exudate cells were incubated with indicated stimulants for 24 h and IL-6 levels in the supernatant was measured by ELISA. (FIG. 50C). Wildtype mice were immunized i.m. with 2.5 pg of ViPS, and phenol extracted ViPS in the absence and presence of Lipid A (5 pg of Kdo2 Lipid A from Avanti Polar Lipids, Alabaster, AL). ViPS-specific IgM and IgG levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparisons test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001;
** = p<0.001; * = p<0.05.
FIGs. 52A-52B illustrate Turbo adjuvanticity under antigen-limiting conditions.
C57BL/6 male and female mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with (A) 2.5, 0.5, 0.1, 0.02 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). (B) Mice immunized with 2.5 pg of Vi PS adjuvanted with Turbo containing indicated amounts of MPLA. Vi PS-specific IgM and IgG3 levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparisons test, and statistically significant differences were indicated as **** = p<0.0001; ** = p<0.001; * = p<0.05.
FIG. 53 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to phenol extracted ViPS in both inbred and outbred mice. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIG. 54 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to ViPS subunit vaccines in both inbred and outbred mice. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIG. 55 illustrates that Turbo enhances/promotes IgM and all four IgG isotype responses to ViPS conjugate vaccine in both inbred and outbred mice using Typbar TCV® vaccines. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05. FIGs. 56A-56B illustrate that Turbo adjuvanticity to unconjugated ViPS vaccine is independent of sex. Male mice are represented in FIG. 56A, while female mice are represented in FIG. 56B. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIGs. 57A-57B illustrate that Turbo adjuvanticity to conjugated ViPS vaccine is independent of sex. Male mice are represented in FIG. 57A. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = pO.OOOl; *** = pO.OOl; ** = p<0.001; * = p<0.05.
FIGs. 58A-58D illustrate Turbo as an adjuvant promotes Vi PS-specific antibody responses to unconjugated Vi PS in the young mice and enhances Vi PS-specific antibody responses to conjugated Vi PS vaccine (Typbar TCV®) in adult, and aged (old) mice. Wildtype (C57BL/6J) (n = 6) male and female mice of indicated ages (in weeks) were immunized i.m. in the thigh of the hind limb with 50 pl of (FIG. 58A) Vi PS or (FIG. 58B) Phenol-extracted ViPS, and (FIGs. 58C& 58D) Typbar TCV® vaccine containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Vi PS-specific IgM and IgG levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparisons test, and statistically significant differences were indicated as **** = p<0.0001; *** = pO.OOl; ** = pO.OOl; * = p<0.05.
FIG. 59 illustrates that the immunogenicity of Meningococcal subunit vaccines is dependent on MyD88, an adaptor for multiple TLR signaling. Wildtype (C57BL/6J) male and female mice of indicated genotype were immunized i.m with 50 pl of (FIG. 59A) Menveoi® (4 micrograms of MenACWY) or (FIG. 59B) MenQuadfi® (2.5 micrograms of MenACWY) or with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Meningococcal serotype-specific IgM and IgG levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = pO.OOOl; *** = p<0.001; ** = p<0.001; * = p<0.05. On day 21 of primary immunization mice were reimmunized as indicated with an arrow.
FIGS. 60A-60B illustrate that a prime-boost strategy with Turbo as an adjuvant enhances antigen-specific response in infant mice and 100% seroconversion. Wildtype (C57BL/6J) male and female mice of 9 days (infant) were immunized subcutaneously with 25 microliters of (FIG. 60A) Menveo® (2 micrograms of MenACWY) or (FIG. 60B) MenQuadfi® (1 .25 micrograms of MenACWY) or with (Black filled circles) and without (white circles) Turbo (containing 2.5 pg of MPLA). Meningococcal serotype-specific IgM and IgG levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05. On day 15 of primary immunization mice were reimmunized as above.
FIGs. 61A-61C illustrate that Turbo as an adjuvant promotes antibody responses to multivalent meningococcal vaccines in young adult and aged mice. Wildtype (C57BL/6J) (n = 8) male and female mice of 9 weeks (young adults) or 102 weeks (aged) were immunized i.m. with 50 pl of (FIGs. 61A & 61C) Menveo® (2.5 micrograms of MenACWY) or (FIG. 61B) MenQuadfi® (4 micrograms of MenACWY) with (Black circles) and without (white circles) Turbo (containing 5 micrograms of MPLA). Meningococcal serotype-specific IgM and IgG levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIG. 62 illustrates that Turbo as an adjuvant promotes antibody responses to H. influenzae PRP-conjugate vaccines Hiberix.
FIG. 63 illustrates that Turbo as an adjuvant promotes antibody responses to Rabies viral vaccine, Imovax.
FIGs. 64A-64D illustrate a comparison of Turbo adjuvanticity with that of Squalene- based emulsion or Alum in the context of bacterial or synthetic polysaccharide, Protein conjugated polysaccharide vaccine or protein antigen. Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with various bacterial polysaccharide (ViPS), protein conjugated- Vi PS vaccine (Typbar TCV), NP-CGG, a commonly used model of protein antigen, or a synthetic polysaccharide NP-Ficoll. (FIG. 64A) 2.5 pg of unconjugated Vi PS with indicated adjuvants (Turbo containing 5 pg of MPLA) or 5% squalene emulsion. (FIG. 64B) Mice immunized with 2.5 pg protein conjugated- Vi PS vaccine (Typbar TCV) adjuvanted with Turbo, Squalene-based emulsion, or Alum (Imject™ Alum Adjuvant at containing 1 mg aluminum hydroxide & 1 mg magnesium hydroxide), (FIG. 64C) NP-CGG adjuvanted with Turbo or Alum; (FIG. 64D) NP-Ficoll adjuvanted with and without Turbo. ViPS-specific antibody (A&B) and NP-specific antibody (C&D) levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparisons test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIGs. 65A-65D illustrate that Turbo as an adjuvant induces durable antibody responses, promotes antibody cl ass- switching to all four IgG isotypes and affinity maturation, and enhanced germinal centers in the draining lymph nodes. Male and female C57BL6 mice (n=6) of 8-10 weeks of age were immunized i. m. in the thigh of the hind limb NP-CGG, a commonly used model of protein antigen, with Turbo, or Alum (Imject™ Alum Adjuvant containing 1 mg aluminum hydroxide & 1 mg magnesium hydroxide). On day 28 mice were boosted with the same antigens in the same area of primary immunization. NP-specific antibody levels were measured by ELISA. The ELISA values obtained with NP30-BSA or NP-18 BSA coated plates measures NP-specific antibodies of diverse affinities, whereas the ELISA values obtained with NP-2 BSA coated plates measures high affinity antibodies. The ratio of NP2/NP18 ELISA values indicate affinity maturation. (FIG. 65A) Turbo-adjuvanted protein antigen shows durable IgG responses of all 4 isotypes. (FIG. 65B) Turbo promotes affinity maturation of all 4 IgG subclasses. (FIGs. 65C & 65D) Represents the size of germinal centers (GC). Draining lymph node on 14 days of primary immunization were sectioned and stained fluorescent labeled GL7, a GC B cell marker and B (IgD) and T (CD4) cell specific antibodies. Immunofluorescent sections were scored as observer blind and statistical differences were determined using Mann-Whitney test. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIGs. 65A-65D illustrate that Turbo as an adjuvant induces durable antibody responses, promotes antibody cl ass- switching to all four IgG isotypes and affinity maturation, and enhanced germinal centers in the draining lymph nodes. Male and female C57BL6 mice (n=6) of 8-10 weeks of age were immunized i. m. in the thigh of the hind limb NP-CGG, a commonly used model of protein antigen, with Turbo, or Alum (Imject™ Alum Adjuvant containigl mg aluminum hydroxide & 1 mg magnesium hydroxide). On day 28 mice were boosted with the same antigens in the same area of primary immunization. NP-specific antibody levels were measured by ELISA. The ELISA values obtained with NP30-BSA or NP-18 BSA coated plates measures NP-specific antibodies of diverse affinities, whereas the ELISA values obtained with NP-2 BSA coated plates measures high affinity antibodies. The ratio of NP2/NP18 ELISA values indicate affinity maturation. (FIG. 65A) Turbo-adjuvanted protein antigen shows durable IgG responses of all 4 isotypes. (FIG. 65B) Turbo promotes affinity maturation of all 4 IgG subclasses. (FIGs. 65C & 65D) Represents the size of germinal centers (GC). Draining lymph node on 14 days of primary immunization were sectioned and stained fluorescent labeled GL7, a GC B cell marker and B (IgD) and T (CD4) cell specific antibodies. Immunofluorescent sections were scored as observer blind and statistical differences were determined using Mann-Whitney test. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = pO.OOOl; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIG. 66A-66B illustrate that Turbo adjuvanticity occurs when it is administered ipsilaterally to the antigen. Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the right thigh of the hind limb with NP-CGG, a commonly used model of protein antigen, or protein conjugated-Vi PS vaccine (Typbar TCV) were bedside mixed without (diamond symbol) or Turbo (Black circles; ipsilateral). In a group of mice (open circles) where the antigen was administered in the right thigh, and the adjuvant was injected in the left thigh (i.e., contralateral). NP-specific antibody (FIG. 66A) and ViPS-specific antibody (FIG. 66B) levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05; N.S. = Not statistically significant.
FIGs. 67A-67B illustrate that Turbo adjuvanticity to unconjugated polysaccharide vaccines is dependent on TLR4 and MyD88. Ten-twelve-week-old mice of indicated genotype on C57BL/6J background of both sexes (n=8-15 mice) were immunized i.m. with (FIG. 67A) 2.5 pg of ViPS or (FIG. 67B) 2.5 pg of phenol -extracted ViPS admixed with Turbo (containing 5 pg MPLA), and ViPS-specific IgM and IgG isotype levels were measured by ELISA. Statistics done using 2-way ANOVA Sidak’s multiple comparison test, and statistically significant differences were indicated as **** = p<0.0001; *** = p<0.001; ** = p<0.001; * = p<0.05.
FIG. 68 illustrates that Turbo-mediated adjuvanticity for polysaccharide vaccines is dependent on co-stimulatory molecules CD40, CD80 and CD86. Wildtype (C57BL/6J) male and female mice of indicated genotype were immunized i.m with 50 pl of phenol extracted ViPS (2.5 micrograms) admixed with Turbo (containing 5 ig of MPLA). ViPS-specific IgM and IgG isotype levels were measured by ELISA.
FIG. 69 illustrates the structures of the TLR4, TLR1/2, and TLR2/6 homodimerizing agonists MPLA, Pan CSIGi, and ParrnC SK i, respectively.
FIG. 70 illustrates the structural basis of lipopolysaccharide recognition by the TLR4- MD-2 complex. Figure is reproduced from Park et al. Nature. (2009) 458, 1191-1195.
FIG. 71 illustrates the recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Figure is reproduced from Kang et al. Immunity. (2009) Dec 18;31 (6): 873-84.
FIG. 72 illustrates the crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Figure is reproduced from Jin et al. Cell. (2007) Sep 21;130(6):1071-82.
FIG. 73 illustrates a comparison of Turbo adjuvant formulation with various TLR phenol extracted ViPS ligands.
FIG. 74 illustrates a comparison of Turbo adjuvant formulation with various TLR Typbar ligands.
FIG. 75 illustrates that Turbo as an adjuvant promotes antibody responses to haptenated protein antigens in all ages.
DETAILED DESCRIPTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. An exemplary disease is a bacterial infection, and associated symptoms.
As used herein, the term “antibody” means whole, intact antibody molecules, as well as fragments of antibody molecules that retain immunogen-binding ability, including the well- known active fragments F(ab')2, and Fab. Antibodies are generated against antigens during the course of an immune response against a pathogen or immunogen and can include IgM and IgG, and all other IgG isotypes.
By "fragment" is meant a portion of a polypeptide, polysaccharide, or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule, polysaccharide, or polypeptide.
By "binding to" a molecule is meant having a physicochemical affinity for that molecule.
By the term “specifically binds,” as used herein, is meant a ligand, which recognizes and binds with a binding partner present in a sample, but which ligand does not substantially recognize or bind other polypeptides in the sample.
By "decreases" is meant a negative alteration of at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.
By "effective amount of1 is meant an amount of an immunogenic composition sufficient to induce or enhance an immune response in a subject. Levels of induced immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by enzyme-linked immunosorbent assay, agglutination assay or any other method known in the art. The effective amount of active compound(s) used to practice the present disclosure for prophylaxis or for therapeutic treatment of a disease varies depends upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living organism is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
By "immune response" is meant the actions taken by a host to defend itself from pathogens or abnormalities. The immune response includes innate (natural) immune responses and adaptive (acquired) immune responses. Innate responses are antigen non-specific. Adaptive immune responses are antigen specific. An immune response in an organism provides protection to the organism against pathogenic infections when compared with an otherwise identical subject to which the composition or cells were not administered or to the human prior to such administration.
"Proliferation" is used herein to refer to the reproduction or multiplication of similar forms, especially of bacterial or eukaryotic cells. That is, proliferation encompasses production of a greater number of bacterial or eukaryotic cells, and can be measured by, among other things, simply counting the numbers of bacterial or eukaryotic cells, measuring incorporation of 3H- thymidine into bacterial or eukaryotic cells, and the like.
A "protective immune response" against an infectious disease (e.g., caused by a bacterial or viral pathogen) refers to an immune response exhibited by a subject e.g., a mammal) that is protective against disease when the individual is subsequently exposed to and/or infected with wild-type bacteria or viruses. Typically, the protective immune response results in detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing bacterial or viruses of the same strain and/or subgroup (and possibly also of a different, nonvaccine strain and/or subgroup) in vitro and in vivo. By "increases" is meant a positive alteration of at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.
By “infectious disease” is meant a disease or condition in a subject caused by a pathogen that is capable of being transmitted or communicated to a non-infected subject. Non-limiting examples of infectious diseases include bacterial infections, viral infections, fungal infections, parasitic infections, and the like.
By “pathogen” is meant an infectious agent, such as a Salmonella enterica serovar Typhi, Salmonella enterica serovar Typhimurium (e. ., S. Typhi, S. Typhimurium), capable of causing infection, producing toxins, and/or causing disease in a host.
As used herein, "sample" or “biological sample” refers to anything, which may contain the cells of interest (e.g, bacteria) for which a screening method or treatment is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Such a sample may include diverse cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cell(s). Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid, or the like.
A “subject” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, primates, livestock, and pets, such as bovine, porcine, ovine, canine, feline, and murine mammals. Preferably, the subject is human.
By “reference” is meant a standard or control condition. A "reference sequence" is a defined sequence used as a basis for sequence comparison.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or improving an infectious disease or condition and/or one or more symptoms associated therewith. It will be appreciated that, although not precluded, treating an infectious disease or condition and/or one or more symptoms associated therewith does not require that the disorder, condition, disease, or symptoms associated therewith be completely ameliorated or eliminated.
By the term "vaccine" as used herein, is meant a composition, a lipid, a polysaccharide, a protein, or a nucleic acid of the disclosure, which serves to protect a subject against an infectious disease (e.g., bacterial disease) and/or to treat a subject having an infectious disease compared with an otherwise identical animal to which the vaccine is not administered or compared with the subject prior to the administration of the vaccine. As used herein, each recitation of the term “Turbo” independently refers to an exemplary non-limiting liposome adjuvant of the disclosure.
By “virulence” is meant a degree of pathogenicity of a given pathogen or the ability of an organism to cause disease in another organism. Virulence refers to an ability to invade a host organism, cause disease, evade an immune response, and produce toxins.
By “virulent” or “pathogenic” is meant a capability of a bacterium to cause a severe disease.
By “non-pathogenic” is meant an inability to cause disease.
By “wildtype” is meant a non-mutated version of a gene, allele, genotype, polypeptide, or phenotype, or a fragment of any of these. It may occur in nature or be produced recombinantly.
As used herein, the term “derived from” encompasses, without limitation: an antigenic molecule that is isolated or obtained directly from an originating source (e.g. a bacterium, a virus, a fungus, a protozoan, or a multicellular parasite); a synthetic or recombinantly generated antigenic molecule that is identical or substantially related to an antigen from an originating source; or an antigenic molecule which is made from an antigen of an originating source or a fragment thereof.
The terms "comprise," "comprises," and "comprising" are open-ended and will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element(s) or step(s). When used herein the term "comprising" can be substituted with the term "containing," "including," or "having."
When used herein "consisting of or “consists of’ excludes any element or step not specified in the claim. When used herein, "consisting essentially of or “consists essentially of’ does not exclude elements or steps that do not materially affect the basic and novel characteristics of the claim.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Description
The present disclosure provides immunogenic compositions useful for the induction of immune responses in subjects against antigens, particularly protein, polysaccharide, and polysaccharide-containing antigens. In some aspects, the immunogenic compositions comprise liposomes comprised of an adjuvant (e.g., monophosphoryl Lipid A (MPLA), Di- or Tri-acyl lipopeptides (Pam2CSK4, and PamsCSK^, and combinations thereof), 1,2-dipalmitoyl-sn- glycero-3 phosphocholine (DPPC), and a protein, polysaccharide, or polysaccharide-containing antigen. In certain embodiments, the compositions can further comprise a surfactant, such as but not limited to polyoxyethylene sorbitan monooleate.
As shown in the Examples, the compositions of the disclosure have unexpected efficacy in inducing antibody immune responses, particularly IgM and IgG based responses, against molecules (e.g., proteins and polysaccharides) which would be poorly immunogenic otherwise.
Compositions
In some embodiments, the disclosure provides immunogenic compositions useful for stimulating immune responses, particularly antibody responses against antigens including proteins, polysaccharides, or polysaccharide-containing antigens. In some embodiments, these compositions induce antibody responses in the absence of T cell help, and involve the stimulation of B cells, particularly Bl type B cells, which mediate long-lasting memory B cell responses. Many bacterial and viral antigens make use of cell-surface glycosylation to aid in target cell infection and avoidance of host immunity. As such, targeting of an antibody response to cell surface polysaccharides and polysaccharide-protein conjugates enables the blocking of infection and immune elimination of pathogenic bacterial cells and viral particles prior to or early in an infection, especially prior to the onset of T cell immunity, which requires priming by antigen presenting cells and antigen-recognition (of infected cells, in the case of CD8+ T cell immunity).
Also provided is an immunogenic composition comprising a liposome or nanosome adjuvant comprising an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof.
MPLA is a derivative of lipid A, the naturally occurring hydrophobic group of the bacterial cell wall component lipopolysaccharide (LPS). LPS covers the surface of most Gramnegative bacteria and is a highly immunogenic molecule, which is recognized by most mammalian immune systems by receptors of the toll-like receptor (TLR) family. In mammals, including humans, the TLR that recognizes LPS is TLR4. It is lipid A that is the immunogenic part of lipopolysaccharide. MPLA is a synthetic structural analog of Lipid A, and is available as a number of derivates including, but not limited to, PHAD® and 3D(6-acyl) PHAD®. In certain embodiments of the current disclosure, the MPLA acts as an activator of immune cells by signaling through TLR4 and other receptors, and enhances the immunogenicity of the antigen by providing a so-called “danger signal” which activates immune cells to secrete inflammatory cytokines, chemokines, and other signaling molecules, all of which combine to enhance the development of an antibody response, including IgM, IgA, and IgG type antibodies against the antigen.
PanuCSh (Pam2CysSerLys4) is a synthetic diacylated lipopeptide (LP) and a potent activator of the pro-inflammatory transcription factor NF -KB. Pan CSIGj mimics the acylated amino terminus of bacterial LPs and is recognized by the TLR2/TLR6 heterodimer. In certain embodiments of the current disclosure, the ParmCSI acts as an activator of immune cells by signaling through TLR2/6 and other receptors, thereby enhancing the immunogenicity of the antigen.
Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide (LP) and a TLR2/TLR1 ligand. It is a potent activator of the pro-inflammatory transcription factor NF-KB. Pam3CSK.4 mimics the acylated amino terminus of bacterial LPs. Bacterial LPs are a family of pro-inflammatory cell wall components found in both Gram-positive and Gram-negative bacteria. The stimulatory activity of these LPs resides in their acylated amino terminus. These bacterial LPs are recognized by TLR2, a receptor that plays a pivotal role in detecting a diverse range of pathogen-associated molecular patterns (PAMPs). At the cell surface, TLR2 forms a heterodimer with co-receptors TLR1 or TLR6, depending upon either tri- or diacylation of the ligand. Once a ligand binds to either TLR2-TLR1 or TLR2-TLR6, a MyD88-dependent activation of NF-KB and AP-1 occurs, ultimately leading to an innate immune response. Recognition of Pam3CSK4, a triacylated LP, is mediated by TLR2 which cooperates with TLR1 through their cytoplasmic domain to induce the signaling cascade leading to the activation of NF-KB.
As polysaccharide antigens are normally poorly immunogenic, the combination with the compositions of the disclosure allows for development of effective humoral immune responses to prevent, ameliorate, and/or treat infection. Due to its inherently immunogenic nature, it is envisioned that any MPLA or Pam2CSK4 or Pam3CSK4 analogue or derivative that retains the ability to be recognized by TLR-family receptors is capable of being used in the immunogenic compositions of the disclosure, including but not limited to MPLA, PHAD®, 3D(6-acyl) PHAD®, 3D-MPLA, KDO DPLA, Pam2CSK4, or Pam3CSK4.
Also provided is an immunogenic composition comprising a liposome or nanosome adjuvant comprising an effective amount of l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof. DPPCs and their analogues or derivatives are amphipathic lipids, with hydrophilic heads, composed of the polar phosphatidylcholine group, and hydrophobic tails, formed by two nonpolar palmitic acid (Cl 6) chains. In the compositions of the disclosure, DPPC or DPPC analogues/derivatives readily and spontaneously form micelles, monolayers, bilayers, liposomes, and nanosomes when in contact with polar solvents, such as water. In this way, DPPC complexes with MPLA or MPLA-derivates or Pam2CSK4 or derivative, or Pam3CSK4 or derivatives to form larger structures encompassing MPLA and the antigens of the disclosure, including the proteins, polysaccharides, or polysaccharide-based molecules of the disclosure such that they are capable of enhancing the interaction with immune cells. In certain embodiments, it is envisioned that any analogue, derivative or variant of DPPC that is capable of forming micelles, monolayers, bilayers, liposomes, and nanosomes in polar solution is capable of being used in the compositions of the disclosure. Examples of such analogues, derivatives, or variants include, but are not limited to l,2-distearoyl-sn-glycero-3- phosphocholine, l,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l -glycerol)], 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (succinyl), l,2-dimyristoyl-sn-glycero-3-phosphate, l,2-dimyristoyl-sn-glycero-3- phosphocholine, l,2-distearoyl-sn-glycero-3-phosphate, l,2-dipalmitoyl-sn-glycero-3 -phosphate, and l,2-dipalmitoyl-sn-glycero-3-phosphocholine, among others. Also provided is an immunogenic composition comprising composition comprising a surfactant such as polyoxyethylene sorbitan monooleate or an analogue or derivative thereof. Also known as polysorbate 80 or tween 80, polyoxyethylene sorbitan monooleate is a nonionic surfactant and emulsifier that is commonly used in the art in a wide variety to pharmaceuticals, foods, and cosmetics. In the compositions of the current disclosure, polysorbate 80 or its analogues or derivatives aid in stabilizing the size of the liposomes or nanosomes which is optimal for interaction with immune cells. Polysorbate 80 or its analogues or derivatives are also known to enhance the cellular ATP release at the site of injection, thus enhancing the immunogenicity of the compositions of the disclosure, and ultimately enhancing the antibody responses generated against the antigenic molecules of the disclosure. It is envisioned that in various embodiments of the disclosure, any polysorbate analogue or derivative molecule can be used in the compositions of the disclosure, including but not limited to polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 65, among others.
Compounds
In other aspects, the present invention provides a compound or any salt, solvate, geometric isomer, or stereoisomer thereof. In some embodiments, the compound is disclosed in FIG. 6. In other embodiments, the compound is disclosed in Table 5, which discloses tripeptides, their corresponding aldehydes, and their corresponding oximes.
In certain embodiments, the compound is at least one of:
MPLA
Figure imgf000026_0001
D(6-Acyl) MPLA
Figure imgf000026_0002
Pam2CSK4 (Pam2CysSerLys4)
Figure imgf000027_0001
or a salt, solvate, geometric isomer, or stereoisomer thereof.
In certain embodiments, the compound is at least one of:
DPPC
Figure imgf000027_0002
or a salt, solvate, geometric isomer, or stereoisomer thereof.
In certain embodiments, the compound is at least one of: Polysorbate 80
Figure imgf000028_0001
or a salt, solvate, geometric isomer, or stereoisomer thereof.
The compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. A compound illustrated herein by the racemic formula further represents either of the two enantiomers or mixtures thereof, or in the case where two or more chiral center are present, all diastereomers or mixtures thereof.
In certain embodiments, the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
In all of the embodiments provided herein, examples of suitable optional substituents are not intended to limit the scope of the disclosure. The compounds of the disclosure may contain any of the substituents, or combinations of substituents, provided herein.
Salts
The compounds described herein may form salts with acids or bases, and such salts are included in the present disclosure. The term "salts" embraces addition salts of free acids or bases that are useful within the methods of the disclosure. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. In certain embodiments, the salts are pharmaceutically acceptable salts. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification, or formulation of compounds useful within the methods of the disclosure.
Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic, 2- hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic, P-hydroxybutyric, salicylic, galactaric, galacturonic acid, glycerophosphonic acids and saccharin (e. , saccharinate, saccharate). Salts may be comprised of a fraction of one, one or more than one molar equivalent of acid or base with respect to any compound of the disclosure.
Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N, N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
Pharmaceutical Compositions and Formulations The disclosure provides pharmaceutical compositions comprising at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, which are useful to practice methods of the disclosure. Such a pharmaceutical composition may consist of at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt, solvate, geometric isomer, or stereoisomer thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In certain embodiments, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous or another route of administration. A composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically based formulations.
In certain embodiments, the compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at 15 least one compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e. ., RECOMB UMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable 20 carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
Formulations may be employed in admixtures with conventional excipients, i.e.. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of 5 administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or fragranceconferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics, or hypnotic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
The composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
The composition may include an antioxidant and a chelating agent which inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g., di sodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total 25 weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
Therapeutic Methods
The methods and compositions provided herein can be used to generate an immune response in a subject against an antigen (e.g., a protein or polysaccharide or polysaccharide- containing antigen). In general, antigens combined with the immunogenic compositions described herein can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an immune response specific for the antigen (e.g., a polysaccharide or polysaccharide-containing antigen).
Also provided are methods of treating, ameliorating, and/or preventing a disease in a subject (e.g., a bacterial disease) comprising immunizing the subject with a composition comprising an antigen (e.g., a protein, a polysaccharide, or polysaccharide-containing antigen) and the immuno-adjuvant of the disclosure.
The methods include administering an immunologically effective amount of an antigen provided herein, and/or an immunologically effective amount of an immunogenic composition provided herein to an individual in a physiologically acceptable carrier. Typically, the carrier or excipient for vaccines provided herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is affected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
In some embodiments, the methods also include administering an additional adjuvant, such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof, with the immunogenic composition of the disclosure. Optionally, a formulation for prophylactic administration also contains one or more adjuvants for enhancing the immune response to the antigens (e.g., polysaccharide or polysaccharide-containing antigens). Suitable adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil, or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59.
Pharmaceutical formulations that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, parenteral, pulmonary, intranasal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, and immunologically based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
The pharmaceutical formulations described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the cells into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
In certain embodiments, the cells of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical formulations of the cells of the disclosure include a therapeutically effective amount of the cells of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
Administration/Dosing
In the clinical settings, delivery systems for the compositions described herein can be introduced into a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical formulation of the composition can be administered by inhalation or systemically, e.g., by intravenous injection.
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the manifestation of symptoms associated with the disease or condition. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the composition of the present disclosure to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or condition in the subject. An effective amount of the composition necessary to achieve a therapeutic effect may vary according to factors such as the extent of implantation; the time of administration; the duration of administration; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder; age, sex, weight, condition, general health, and prior medical history of the subject being treated; and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the composition without undue experimentation. Actual dosage levels of the cells in the pharmaceutical formulations of this disclosure may be varied so as to obtain an amount of the composition that are effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
Routes of Administration
Routes of administration of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e. ., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable formulation of the composition sand dosages includes, for example, dispersions, suspensions, solutions, beads, pellets, magmas, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, aerosolized formulations for inhalation, compositions, and formulations for intravesical administration and the like.
It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, differentiation methods, engineered tissues, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
Kits
The disclosure provides kits for the treatment or prevention of a disease and/or disorder, such as but not limited to a bacterial infection. In certain embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of an immunogenic composition (e.g., nanosomes or liposomes comprising Monophosphoryl Lipid A and 1,2- dipalmitoyl-sn-glycero-3 phosphocholine with a polyoxyethylene sorbitan monooleate surfactant) in unit dosage form. In some embodiments, the kit comprises a device (e.g., nebulizer, metered-dose inhaler) for immunogenic composition dispersal or a sterile container which contains a therapeutic or prophylactic immunogenic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired an immunogenic composition of the disclosure is provided together with instructions for administering the immunogenic composition to a subject having or at risk of contracting or developing a bacterial or viral infection. The instructions will generally include information about the use of the composition for the treatment or prevention of a bacterial or viral infection. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of a bacterial or viral infection or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. EXPERIMENTAL EXAMPLES
The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples, therefore, specifically point out embodiments of the present disclosure, and are not to be construed as limiting in any way.
Formulation and manufacturing of Turbo
1. To the 1.0 mg MPLA or Pai CSB or Pan CSBU glass vial add 83 mL of 10 mg/mL DPPC. The molar ratio is MPLA/Pan CSK PamsCSK^ 1: DPPC 2.
2. To make sure the 83 mL is in touch with the lyophilized MPLA, add 417 mL of chloroform. Vortex the solution vigorously.
3. Transfer the 500 ml of MPLA/Pan^CSKV PamaCSK4 1 : DPPC 2 mixture into a 5 ml polypropylene plastic tube.
4. Leave the plastic tube open for overnight in the fume hood.
5. Once the whole material is dry, add 2.0 mL of 1% Tween 80 containing water and let it sit for -10-15 minutes.
6. Sonicate for 5 times (30 secs each time).
7. Pool the two 2 ml sonicates as 4 ml using a 22G needle hooked to a 5 ml syringe. Replace the needle with a 25G emulsifying steel bar-enforced syringe needle hooked to the 5 ml syringe.
8. After hooking the other end of the emulsifying steel bar-enforced needle to another 5 ml syringe, make 40-60 passes.
9. Subject the emulsified solution to filtration 5 times with 0.22 mm PES synthetic filter (Millipore).
10. Transfer the -3.5 ml of the material to sterile Eppendorf tubes and store at 4°C. 11. Nanosight analysis is required to confirm the particle size and homogeneity. It is expected to be 150+/-50 nm)
12. Turbo is ready to be mixed with the ViPS or other antigens for immunization.
Example 1: Characterization of an optimal B cell response.
Using the experimental model of Borrelia hermsii bacteremia, studies determined a role for Bib type B cells, a subset of mature B cells which mediate long-lasting memory responses, especially the development of antigen-specific IgM and IgG responses, in the absence of T cellhelp (FIG. 1). Specifically, these studies found that Bib cells can expand concurrently with the resolution of bacteremia and persist for long time periods (FIG. 2) using Borrelia hermsii infection as a model. Here, Bib cells from convalescent mice but not from naive mice generated a specific antibody response and conferred long-lasting immunity, indicating that the protective immune response corresponds to Bib cell expansion and persistence as in the case of conventional B cell memory.
B cell responses to T cell-independent antigens such as bacterial polysaccharides are generated primarily by cross-linking B cell antigen receptors (BCR). Although Pneumococcal polysaccharide is also recognized by Bib cells, it does not induce antibody responses in X-linked immunodeficient mice (xid) mice, which have a mutation in the gene encoding for Bruton’s tyrosine kinase (Btk), which is required for optimal BCR-mediated signaling (illustrated in FIG. 3). In contrast, B. hermsii induces not only a specific antibody response but also a selective expansion of Bib cells in xid mice (FIG.2). These expanded Bib cells persist for long time periods, conferring upon the convalescent xid mice resistance to re-infection. These data further revealed that immunostimulatory mechanisms other than BCR signaling can play an important role in the generation and maintenance of T cell independent Bib cell memory. Subsequent studies found that B. hermsii can activate Toll-like receptors (TLRs) (illustrated in FIG. 4) and that mice deficient in both Btk and MyD88, an adaptor protein required by multiple members of the TLR family, are severely impaired in mounting protective responses indicating that a coordinated signaling through BCR and TLRs is critical (FIGs. 6A-6B).
Example 2: Development of Turbo adjuvant for polysaccharide vaccines. Bib cells generate antibody responses to a variety of bacterial antigens, including pneumococcal polysaccharide and the Vi polysaccharide (ViPS) of Salmonella Typhi, the causative agent of Typhoid in humans. Many polysaccharide subunits vaccines lack adjuvants. A few contain aluminum-based salts (Alum). None of the current ViPS subunit vaccines contain an adjuvant (FIG. 29). The recognition that stimulation of both TLR and BCR signaling could result in the generation of robust antibody responses against B. hermsii antigens that was relatively long-lived in mouse models suggest the development of an immuno-adjuvant that can effectively present antigens such that optimal Bib responses are induced. This adjuvant would be uniquely suited for plain polysaccharides and protein-conjugated polysaccharide vaccines, which would be present on a wide variety of bacterial and enveloped viral pathogens. The potential to develop truly effective polysaccharide vaccines is especially relevant because they do not generate long- lasting immune responses despite multiple booster immunizations (FIGs. 7-8). Moreover, more traditional alum-based adjuvants, which are well known in the art, induce a modest 2-fold increase in antibody responses whether or not the polysaccharide antigen is conjugated to a carrier protein (FIG. 9). In fact, the data of the current disclosure show that the adjuvant formulation developed and disclosed herein (Turbo) induces a better response to ViPS conjugate vaccine compared to that induced by alum (FIG. 22).
Optimally, such an immune adjuvant would combine multiple immune signals to generate an optimal immediate antibody response that also provided long-lived immunity. These signals are illustrated in FIG. 28 and include initiation of the response via TLR4 signaling.
MPLA activates TLR4-MyD88 axis to induce pro IL-la and pro IL-10 (Signal 1). The TLR4-Trif axis (Signal 2) results in IRF3-mediated IFNy expression. The IFNy signaling via IFNR (Signal 3) is required for the induction Caspase 11. In macrophages MPLA alone does not induce activation of Caspase 1 or IL- 10 secretion and requires activation of inflammasome such as NLRP3. In support of this, we have not seen an impairment in Turbo adjuvanticity in NLRP3- or Caspl -deficient mice (FIG. 23). Therefore, a role for IL-10 is unlikely in Turbo adjuvanticity. Since Caspase 11 is a sensor for LPS (Signal 4) in the cytoplasm, and Turbo formulation as liposomes enable an efficient intracellular transport of MPLA, we hypothesize that the MPLA-mediated adjuvanticity in Turbo formulation drives Caspase 11-dependent IL-la maturation and release. This possibility is supported by the fact that IL-la is activated by caspase- 11 by cleavage at a conserved site and the subsequent secretion of IL-la from macrophages. MyD88 /_ mice are significantly impaired for Turbo adjuvanticity (FIG. 24 and 39). MyD88 is also required for IL-1R1 signaling, it is possible that IL-la-IL-lRl-MyD88 signaling axis may be involved in B cells, in addition to MPLA-TLR4-MyD88 axis. These axes are expected to synergize with BCR signaling in antigen-specific B cells to upregulate B cell survival receptor BAFFR, plasma cell-differentiation receptor, TACI, and plOO, a precursor of NF-KB2. NF-KB2 is central for CD40L-CD40-mediated expression of AID in B cells. NF-KB2 is also required for BAFF-BAFFR-mediated survival of B cells through the expression of prosurvival molecules Mcl-1 & Bcl-XL. Since an efficient class-switching is occurring from IgM to all the 4 IgG subclasses and this process required AID, we hypothesize those co-stimulatory signals generated from TLR4/MyD88 axis in Dendritic cells or macrophages play a role in antigen-specific B cell responses.
One of the core components of the adjuvant is monophosphoryl lipid A (MPLA) (FIG. 13). While Lipid A is naturally the hydrophobic group of the bacterial cell wall component lipopolysaccharide, which covers the surface of most Gram-negative bacteria, it is lipid A that is the immunogenic part of lipopolysaccharide. MPLA is a synthetic structural analog of Lipid A, with one phosphate group instead of two (Lipid A has two phosphate groups) (FIG. 13) and is available commercially as PHAD® and 3D(6-acyl) PHAD®. While these molecules are well known as immuno-adjuvants useful for inducing CD4 and CD8 T cells responses, in the adjuvant of the current disclosure, the MPLA of the current disclosure is combined with other molecules useful for specifically stimulating B cell responses, especially given that most T cells recognize peptide-based antigens as presented in the context of HLA or MHC complexes.
As an immunoadjuvant, MPLA has several beneficial effects on adaptive immune responses, including antibody responses. MPLA induces IgM, IgGl & IgG2c to Ova immunization as effectively as Alum or E6020 and also induces CD4 and CD8 responses comparable to that induced by LPS. Shingrix (VZV), Fendrix (HepB) and Cervarix (HPV) vaccines, which contain MPL® adjuvant in the forms of AS01 (3D6A MPLA from S. minn+QS21 Saponin) and AS04 (50 ug 3D6A MPLA+ Alum), have been found to have clinical efficacy despite the fact that 3D6A MPLA activates mouse TLR4 more efficiently that human TLR4 activation and 3D6A MPLA is 50 times less potent than MPLA. Lastly, MPLA alone does not induce IL-ip, but does in the presence of Alum or QS-21 in a caspase 1/ 11 -dependent fashion from mBMDMs or iMOs. Interestingly, MPLA+QS21 -mediated response requires ASC, MyD88, Trif, TLR4, NLRP3 but not NLRC4. The IL-18 production is also dependent on NLRP3.
Practically, MPLA offers several advantages for use in a clinical-grade immuno-adjuvant. First, as a synthetic TLR4 agonist, MPLA can be manufactured in a cGMP facility with high purity (>99%), improved stability and reduced manufacturing cost. Also, the lack of systemic toxicity and ability to manufacture it free of contamination by other bacterial components makes it amenable to a variety of adjuvant formulations.
To formulate the immunoadjuvants of the present disclosure, MPLA was combined with l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC), an amphipathic lipid, with a hydrophilic head, composed of the polar phosphatidylcholine group, and hydrophobic tails, formed by two nonpolar palmitic acid (Cl 6) chains. DPPC readily and spontaneously form micelles, monolayers, bilayers, and liposome when it is in contact with a polar solvent, such as water. DPPC is also the main constituent of lung surfactants, reduces the surface tension of the alveolar liquid.
While MPLA/DPPC form the liposome component of the immuno-adjuvant of the present disclosure, the final component is a surfactant which stabilizes the liposomes. As a nonlimiting example, these studies used polyoxyethylene sorbitan monooleate or polysorbate 80. It was found that when used as a surfactant at a concentration above 0.5%, polysorbate 80 helps the stability of the size of the nanosomes/liposomes and enhances the cellular ATP release at the site of injection. Clinically, polysorbate 80 is already in use as an excipient in a majority of FDA- approved vaccines including for influenza (1.2 mg) (FIG. 14).
This immunoadjuvant combination of MPLA and DPPC with polysorbate 80 as a surfactant was dubbed “Turbo” (FIG. 14A). Subsequence studies were conducted in mice in combination with various polysaccharide and polysaccharide-protein conjugates in order to determine the efficacy by which it can induce durable antibody immune responses.
Example 3: In vivo use of Turbo to induce immune responses in mice.
In order to determine the efficacy of Turbo as an immuno-adjuvant, a series of mouse experiments were setup wherein Turbo would be combined with a model polysaccharide antigen, against which humeral immune response could be easily measured by sampling peripheral blood of subject animals. For these studies, Vi polysaccharide (ViPS), ViPS conjugated to Tetanus toxoid (a WHO preapproved vaccine against typhoid), Haptenated Ficoll and haptenated Chicken gamma globulins were used as the antigens (FIG. 15). ViPS has a molecular formula of CioHieNOs and a molecular mass of 278.235. ViPS consists of 600-10000 repeating units, and its O acetylation is about 95%. ViPS has been well characterized as a protective antigen and as a virulence factor. This is the principal immunogen in all typhoid subunit vaccines, which do not contain adjuvants. In fact, studies have indicated that contaminated LPS is likely to account for the immunogenicity of these vaccines (FIGs. 10, 11, 12, 49, 50 and 51).
FIG. 26 or 52 illustrates an example of one such mouse study wherein each animal received 3.4x107 nanosomes (containing 5 ug MPLA) with 0.02 - 2.5 ug ViPS in a total volume of 50 ul delivered intramuscularly. In some animals, a second booster dose was administered more than 28 days after the first immunization. Peripheral blood was drawn at various timepoints in order to assess the concentration of specific antibody generated. Ideally, the use of Turbo/ViPS would integrate various immune signals to induce activation of macrophages and dendritic cells (FIG. 28) as well as stimulating the activation of B cells by dendritic cells in B cell follicles or in the extrafollicular areas of the subject’s lymph nodes (FIG. 28).
Several of these studies also looked at the combination of Turbo/ViPS vaccination with a squalene-based adjuvant. When combined with surfactants, squalene is a common ingredient of commonly used vaccines, in that it stimulates the production of CD4 memory cells. In combination with Turbo/ViPS however, a squalene-based adjuvant (SE; similar to adjuvant MF59) negatively impacted Turbo adjuvanticity in that the combination resulted in significantly lower concentrations of IgM and IgG (FIGs. 20A-20B). When combined with a booster, however, SE did result in greater IgG production, though Turbo alone was better in inducing IgG responses (FIGs. 25B).
A series of follow-up studies was then performed in order to determine the effect of Turbo dosage on the induction of anti-ViPS IgG responses. Here mice received a number of doses ranging from 10 ug, 5 ug, 2.5 ug, and 1.25 ug of MPLA. Results showed observable but not significant correlations between antigen concentration of IgG down to 1.25 ug (FIG. 26A), the use of Turbo resulted in significant increases in IgG when used with even lower doses of antigen (FIG. 26B). These data suggested that Turbo was capable of inducing IgG responses even at sub-optimal antigen doses. A series of studies was then conducted to see whether the inclusion of 2% Tween 80 or polysorbate 80 had any effect on the generation of antibody responses. These data demonstrated that the presence or absence of polysorbate 80 had little effect on the overall generation of ViPS specific IgM and IgG (FIGs. 27A-27B).
Studies also assessed whether a variant of MPLA, 3D6A MPLA had any effect on Turbo efficacy. At least in studies using a single immunization, results showed that while the 3D6A MPLA Turbo group had less IgM concentration at day 7 post vaccine the overall IgG response was comparable to MPLA groups up to 28 days post-immunization (FIGs. 21 & 22).
The use of polysaccharide-based vaccines is of particular interest in pediatric patients, especially against meningococcal and pneumococcal diseases. In order to model pediatric use, a series of mouse studies was conducted using single immunizations in relatively young (3 weeks) mice. The results from these studies showed that while IgM generation was comparably low in these animals regardless of Turbo use (FIG. 18A), the Turbo treated group demonstrated significantly higher concentrations of specific IgG up to 90 days post-vaccination (FIG. 18B). As a test of effective of this treatment as a prophylactic vaccine, animals were then challenged with . typhimurium strain RC60. In this case, vaccination with ViPS alone no significant effect on the amount of bacterial in the blood (as measured by CFU/pl), however the ViPS + Turbo group was found to have significantly lower numbers of bacteria. Together, these data demonstrated that IgG generated by Turbo vaccination could counteract a subsequent bacterial challenge. It was also found that Turbo shows adjuvanticity in an infant (10 day old) mouse immunization model with ViPS-TT conjugate vaccine (FIG. 19).
Previous studies had suggested the importance of TLR-mediated signaling in generating effective antibody responses. In order to determine the effect of TLR4 signaling on the effectiveness of Turbo adjuvant function, a series of similar ViPS vaccination studies were done in TLR4, MyD88, and Trif knockout mice. FIGs. 24A-24B illustrate conclusively that Turbodriven antibody responses are dependent on TLR4-MyD88 axis, as the ablation of either of these genes resulted in dramatically ameliorating the antibody response to vaccination.
The importance of caspase 1 and other components of the NLRP3 inflammasome in the induction of antibodies in rection to Turbo vaccination was then assessed. Here the amount of antibody produced by wildtype or mice deficient in NLPR3, Caspl, or GsdmD as assessed, and results found that Turbo driven antibody responses are not dependent on NLRP3 inflammasome or pyropotosis (FIGs. 23A-23B). Alum adjuvanticity is dependent on NLRP3 inflammasome. The data in FIG. 23 indicates that Turbo adjuvanticity is distinct from that induced by Alum. Indeed, these show that Turbo promotes all IgG isotypes (FIGs. 16 and 17), unlike Alum which shows a skewed response towards IgGl isotype (FIG. 17)
Because the previously presented studies were conducted exclusively in C57BL6 mice, a follow-up study sought to determine whether the dramatic effect of Turbo vaccination was due to a strain-specific factor, as strain to strain variability is known to exit, particularly in aspects of immunity including T cell response skewing (Thl vs Th2). Similar studies as those described previously were conducted in BALB/c and 129Sv mice (FIGs. 32). Results of these studies demonstrated that the effect of Turbo vaccination was independent of strain and sex suggested its benefit in any mammalian system (FIGs. 16, 53, 54 55, 56 and 57). Similar studies were also conducted using outbred strains of mice. Here, mice were immunized with unconjugated ViPS vaccine (obtained from FDA) without and with Turbo and ViPS-specific antibody responses were measured. Results demonstrated that Turbo enhances antibody responses to both unconjugated (FIG. 54) and conjugated (ViPS-TT) vaccine (FIG. 55) in both inbred and outbred mice.
Next, a series of studies was then conducted to examine whether the efficacy shown by Turbo could be found using antigens which were free of LPS contamination. Importantly, ViPS antigen is isolated from bacteria, and therefore, the ViPS preparations used in these studies is contaminated with bacterial components such as LPS. To get a general idea of the amount of LPS contamination present in ViPS preparations, ViPS from several sources was obtained and compared to LPS in mouse peritoneal macrophage activation studies. The ViPS preparations compared were ViPS from Sanofi Pasteur, which was isolated from S. Typhi strain Ty2, ViPS from the US FDA, which was isolated from Citrobacter freundii strain WR7011, and ViPS from International Vaccine Institute, South Korea, which was isolated from S. Typhi, clinical isolate C6524. FIG. 10 shows the results of these studies, which estimate that 2.5 ug of the ViPS dose given to mice as an immunogen is contaminated with ~2 ng of LPS.
In order to determine whether contaminating TLR ligands such as LPS, contribute to the immunogenicity of ViPS vaccines, studies were conducted which compared antibody responses in wildtype and TLR4-deficient mice, which cannot respond to LPS. These studies found that mice deficient in TLR4 are impaired in responding to both plain ViPS vaccine (obtained from US FDA, lot 5) as well as WHO approved ViPS-TT conjugate vaccine (TypBar TCV from Bharat Biotech, India) (FIGs. 11 and 50B). Likewise, MyD88 knockout mice were found to exhibit a much lower response to another unconjugated ViPS vaccine (Typhim Vi), suggesting that this formulation contains contaminants that activate the TLR-related MyD88 pathway (FIG. 46).
In order to eliminate LPS from the studies, ViPS (obtained from US FDA) was subjected to a phenol extraction protocol to remove as much LPS as possible prior to use in mouse vaccination studies. FIGs. 12A and 12B demonstrate that while extracted ViPS is poorly immunogenic, the combination with Turbo can enhance immunogenicity with no difference in IgG levels between extracted and un-extracted Turbo groups. These data suggested that the presence of contaminating LPS in the polysaccharide does not appreciably contribute to the efficacy of Turbo as an immuno-adjuvant. As an additional proof of this observation, a similar study was conducted using the purely synthetic polysaccharide antigen NP-Ficoll. This polysaccharide is not bacterially derived and therefore does not have any amount of contaminating LPS. Similar to previous studies, NP-Ficoll is poorly immunogenic by itself, and does not induce a robust IgG2b or IgG2c response but can stimulate robust IgG responses when combined with Turbo (FIGs. 17A).
To test whether Turbo also promotes antibody responses to protein antigens, a group of mice was immunized with NP conjugated to Chicken Gamma Globulin (NP-CGG), a model antigen for studying T cell-dependent B cell responses. Results found that Turbo promoted class switching to all four IgG isotypes (FIG. 17B). Unlike Alum which activate NLPR3 inflammasome and polarizes antibody response towards IgGl (FIG. 17B), Turbo promoted IgGl, IgG2b, IgG2c/IgG2a, and IgG3, suggesting Turbo adjuvanticity is mediated by a pathway distinct from alum.
Example 4: Turbo induces robust immunity at all ages
The effects of Turbo adjuvant use on mice of varying ages were determined. These studies are of particular importance given the need for pediatric vaccines, especially against pathogens expressing polysaccharide and protein antigens.
FIGs. 30A-30C demonstrate the use of Turbo adjuvant in infant mice (10 days old) vaccinated with a ViPS tetanus toxoid conjugate antigen (ViPS-TT). Over the first 28 days, IgG levels in Turbo vaccinated mice were significantly higher than ViPS-TT antigen alone, and IgM levels were modestly increased (FIG. 30A). Animals were then followed out to 120 days, during which the significant increase in IgG levels in turbo-vaccinated mice continued (FIG. 30B). At the end of the experiments, mice were challenged with S. typhimurhmt strain RC60 bacteria and the persistence of live bacteria was assessed in blood, liver, and spleen. Turbo-vaccinated animals consistently demonstrated lower levels of bacteria (Fig 30C).
A similar study was then conducted in adult mice, which were 2-4 months old. Similar to the experiments conducted with newborn mice, Turbo adjuvant resulted in higher levels of IgM over the first 7 days in the adult mice, followed by significantly higher IgG production over 90 days post-vaccination (FIG. 31). A subsequent study demonstrated that Turbo promotes the production of all IgG isotypes in adult mice when paired with a ViPS-TT conjugate vaccine (FIG. 32). Similar findings were demonstrated in aged mice, which were at least 1.6 years old at the time of vaccination (FIGs. 33 and 58). This aspect was also demonstrated using NP-CGG, haptenated protein antigen (Fig.75). Together, and without wishing to be bound by any particular theory, these data demonstrate the utility of Turbo as an adjuvant with vaccines for all ages of subjects including the very young and aged.
A series of experiments were then performed to determine whether the increased IgG responses observed in reaction to Turbo vaccines could lead to enhanced immune reactions to booster immunization. FIG. 34 shows that Turbo vaccinated and boosted mice produced significantly more IgG as compared to mice receiving an alum-adj uvanted vaccine. The beneficial effects of Turbo over alum were observed across multiple types of IgG, including IgGl, IgG2b, IgG2c, and IgG3 (FIGs. 35A-35B and 65A-65D).
The ability of Turbo adjuvant to synergize with hapten-based vaccines in a pediatric model was also examined. Here, a hapten conjugated NP-CGG antigen was combined with either Turbo or alum and delivered to 10-day old infant mice. Similar to previous studies, while Turbo stimulated a modest increase in IgM over alum at 7 days, the differences between these two adjuvants was not significant over 28 days. IgG production, on the other hand, was significantly higher in Turbo vaccinated mice (FIG. 36).
Example 5: Turbo enhances two commercially available meningococcal vaccines The ability of Turbo to enhance antibody responses to two commonly used meningococcal vaccines, Menveo® and MenQuadfi®, was then assessed. Mice were immunized with two approved meningococcal polysaccharide vaccines (i.e., Menveo® from GSK and MenQuadfi® from Sanofi Pasteur). These vaccines are multivalent, and contain four serotype polysaccharides, namely A, C, W and Y. The results of these studies demonstrated that Turbo enhances both IgM (FIGs. 43 and 61) and IgG (FIGs. 44, and 61) antibody responses to both vaccines and all four serotypes with a single immunization.
Example 6: Mechanisms of Turbo-enhanced immunity
The efficacy of Turbo to enhance antibody responses to antigens, especially IgG responses, raised the question as to the signaling and immunological mechanisms by which it exerts its effects. A series of experiments were then performed to determine which immunological pathways were enhanced by Turbo usage.
As an initial study, Turbo was combined with a number of TLR4 agonists to determine which could combine with Turbo to promote greater antibody production to both tetanus toxoid conjugated and unconjugated ViPS antigen. FIGs. 37A and 37B.
The effect of Turbo on NLRP3 inflammasome and pyroptosis mechanisms was then examined (FIGs. 38A-38B). Here mice lacking expression of one of the components of these mechanisms were vaccinated with Turbo adjuvanted ViPS or ViPS-TT antigens. Results demonstrated similar trends in IgM and IgG production in all mice, suggesting that the adjuvant function of Turbo does is independent of these mechanisms.
Having observed that Turbo can combine with TLR4 agonists, a series of experiments were then conducted in order to demonstrate that Turbo functions by stimulating enhanced signaling trough TLR4 associated pathways. Mice lacking TLR4, MyD88, Trif, or Caspase 1x11 were vaccinated with Turbo/ViPS-TT conjugate vaccines. The results demonstrated that the lack of any of these signaling pathways resulted in lower IgG and IgM production (FIG. 39).
Further, the role of various antigen-presenting cells in Turbo enhancement of antibody responses was assessed. Vaccination with Turbo adjuvanted ViPS and ViPS-TT vaccines was carried out in wildtype or batf3 knockout mice, which lack conventional DC function. Knockout mice demonstrated no loss in IgM or IgG production, and in some cases had higher IgG2c and IgGl expression than wildtype mice, indicating that Turbo does not exert its beneficial effects though this type of antigen presenting cell (FIG. 40). Similar results were observed with the use of RB68C5 antibody, which depletes neutrophils, suggesting that Turbo does not exert its effects through this cell type either (Fig 41 A). Moreover, chlodronate liposomes were used to deplete macrophages before Turbo ViPS/ViPS-TT vaccination. Here, depletion of macrophages significantly decreased IgG production in treated animals (FIG 4 IB). These results demonstrate that the benefits of Turbo adjuvant treatment are exerted via macrophages to enhance IgM and IgG antibody production by B cells. A schematic of these interactions is illustrated in FIG. 42.
Subsequent studies then sought to examine the role of Trif signaling in the enhanced antibody responses generated by Turbo. Here, wildtype, Trif knockout, or Caspase 1x11 knockout mice were immunized with WHO pre-approved typhoid conjugate vaccine (i.e., ViPS- TT, TypBar TCV). (FIG. 45). Results of these studies demonstrated that Turbo adjuvanticity requires signaling through Trif.
A series of studies was then conducted to examine the ability of Turbo to enhance immune responses to unconjugated vaccines. FIGs. 46 and 50 shows that Turbo enhances antibody response to the unconjugated ViPS vaccine Typhim Vi.
Differences of the immune responses to Turbo in inbred and outbred strains of mice were then conducted to see if the antibody responses in these two types of mice would be equivalently robust. FIG. 47 demonstrates that Turbo enhances antibody response to unconjugated ViPS vaccine in both inbred and outbred mice and FIG. 48 illustrates that Turbo likewise enhances antibody response to conjugated ViPS vaccine (ViPS-TT) in both inbred and outbred mice.
To further characterize the TLR signaling of available polysaccharide vaccines, the ability of typhoid Vi Polysaccharide subunit vaccines Typhim Vi® and Typbar TCV® to stimulate mouse and human Toll-Like Receptor 4 (TLR4) was then examined (FIG. 49). HEK293 cells expressing a specific TLR or NOD-like receptor (NLR) with an NF-KB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, were incubated with Typhim Vi® (5 pg ViPS/ml l/5th of human dose of lot V2A451M from Sanofi Pasteur), Typbar TCV® (5 pg ViPS/ml l/5th of human dose of lot 76B21035A from Bharat Biotech), and Vi PS (33 pg ViPS/ml lot 5 PDML158299 from US FDA). Positive controls were 108 heat-killed Listeria monocytogenes cells/ml for m/hTLR2, Poly(I:C) HMW at 1 pg/m for m/hTLR3, E. coli K12 LPS at 100 ng/ml for m/hTLR4, S. typhimurium flagellin at 100 ng/ml m/hTLR5, CL307 at 1 pg/ml m/hTLR7: TL8-506 at 1 pg/ml for hTLR8, TL8-506 at 10 pg/ml for mTLR8, CpG ODN 2006 at 10 pg/ml for hTLR9, CpG ODN 1826 at 1 pg/ml for mTLR9, C12-iE-DAP at 10 pg/ml for m/hNODl, and L18-MDP at 1 pg/ml m/hNODl. HEK -Blue Null cell lines (which do not express any TLR or NLR) incubated with the above agonists served as negative controls. The media containing HEK-Blue Detection™, is designed for the detection of NF-KB induced SEAP expression. After a 16-24-hour incubation the optical density (OD) is read at 650 nm. Results demonstrated that all three vaccine types strongly stimulated human and mouse TLR4, and ViPS also demonstrated significant stimulation of mouse TLR2.
Having observed a key role for TLR4, subsequent studies then examined the effects of eliminating TLR4 signaling. Here, wildtype (C57BL/6J) or TLR4-/- mice 8-10-week-old of both sexes (n=6) were immunized i.m. with 50 pl 2.5 pg of ViPS (lot 5 PDML158299 from US FDA), and ViPS-specific IgM and IgG levels were measured by ELISA (FIG. 51 A). (FIG. 5 IB) Peritoneal exudate cells were incubated with indicated stimulants for 24 h and IL-6 levels in the supernatant was measured by ELISA. (FIG. 51C). Wildtype mice were immunized i.m. with 2.5 pg of ViPS, and phenol extracted ViPS in the absence and presence of Lipid A (5 pg of Kdo2 Lipid A from Avanti Polar Lipids, Alabaster, AL). ViPS-specific IgM and IgG levels were measured by ELISA. Results demonstrated a significant loss of immunogenicity when TLR4 signaling is abrogated.
The immune-enhance effects of Turbo were then observed under antigen-limiting conditions. C57BL/6 male and female mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with (A) 2.5, 0.5, 0.1, 0.02 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). (B) Mice immunized with 2.5 pg of Vi PS adjuvanted with Turbo containing indicated amounts of MPLA. Vi PS-specific IgM and IgG3 levels were measured by ELISA. Data from these studies demonstrated that Turbo resulted in a significant adjuvanticity effect even down to 0.02 pg of antigen and MPLA doses down to 1.25 Pg-
Given that genetically-identical laboratory inbred mouse strains may not necessarily accurately the effect of immune-adjuvants when used in genetically diverse populations of subjects, such as humans, a series of studies then sought to determine whether Turbo is able to promote IgM and all four IgG isotype responses to phenol-extracted ViPS in both inbred (C57BL/6J and 129Sl/SvImJ) and outbred (A/J and CD-I) strains of mice. FIG. 53. Male and female mice of indicated strains of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of Phenol -extracted Vi PS containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
A similar study was performed using Vi Polysaccharide vaccine (FIG. 54). Here male and female mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
And a third set of studies used Tybar TCV (FIG. 55). Male and female mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of Typbar TCV® vaccines containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA.
All three sets of studies demonstrated that Turbo strongly enhanced the immunogenicity of all polysaccharide antigens tested in both inbred and outbred mice.
The possible effects of sex on Turbo efficacy with both conjugated and unconjugated antigens was then examined (Figs 56 and 57). Here, male and female mice of indicated strains of mice of 8-10 weeks of age were immunized i.m. in the thigh of the hind limb with 50 pl of either Typhim Vi vaccine or Typbar TCV vaccine containing 2.5 pg of Vi PS with (Black circles) and without (white circles) Turbo (containing 5 pg of MPLA). Vi PS-specific IgM, IgGl, IgG2b, IgG2a/c, and IgG3 levels were measured by ELISA. Both studies found robust Turbo efficacy in both male and female mice.
The role of TLR4 and MyD88 signaling in the immunogenicity of Menginococcal subunit vaccines was then examined (FIG. 59). Wildtype (C57BL/6J) male and female mice of TLR4-/- and MyD88-/- genotype were immunized i.m with 50 pl of Menveo® (4 micrograms of MenACWY) or MenQuadfi® (2.5 micrograms of MenACWY) or with and Turbo (containing 5 pg of MPLA). Meningococcal serotype-specific IgM and IgG levels were then measured by ELISA and the results demonstrated that immunogenicity is dependent largely on the function of MyD88, rather than TLR4 specifically. MyD88 is an adaptor protein that functions in the signaling through multiple TLR family receptors. One significant potential clinical use of Turbo adjuvanted vaccines would be in the vaccination of the very young. In order to determine the efficacy of a prime-boost strategy with Turbo as an adjuvant in infants, wildtype (C57BL/6J) male and female mice 9 days of age (infant) were immunized subcutaneously with 25 microliters of Menveo® (2 micrograms of MenACWY) or MenQuadfi® (1.25 micrograms of MenACWY) or with and without Turbo (containing 2.5 pg of MPLA) (FIG. 60). Meningococcal serotype-specific IgM and IgG levels were then measured by ELISA. These studies demonstrated that Turbo enhances antigen-specific response in infant mice and 100% seroconversion.
Next, the signaling dependence of Turbo adjuvanted, unconjugated vaccines were then assessed in the context of TLR4 and MyD88 signaling (FIG. 67). Ten-twelve-week-old mice of each genotype (wildtype, TLR4-/-, MyD88-/-, and Trif-/-) on C57BL/6J background of both sexes (n=8-15 mice) were immunized i.m. with 2.5 pg of ViPS or 2.5 pg of phenol-extracted ViPS admixed with Turbo (containing 5 pg MPLA), and ViPS-specific IgM and IgG isotype levels were measured by ELISA. The resulting data revealed that the knockout of TLR4 and MyD88 had the most significant deleterious effect on IgM and IgG production, while the loss of Trif signaling had little effect as compared to wildtype mice.
Having highlighted the key role played by TLR4 and MyD88 in the adjuvant effects of Turbo, subsequent studies then focused on other co-stimulator molecules expressed by antigen presenting cells (APCs), B-cells, and T-cells. Here phenol extracted ViPS (2.5 micrograms) admixed with Turbo was administered to mice lacking CD40, CD80, or CD86 expression. The loss of each gene expression resulted in a significant loss of Turbo efficacy, especially CD86 and CD40, highlighting the importance of the normal functions of the cells expressing these molecules in stimulating enhanced immune responses to vaccines and other antigens when administered with Turbo adjuvant (Fig 68).
Lastly, a series of studies was undertaken to study whether the use of other TLR stimulating agents could lend efficacy to Turbo adjuvant preparations, as previous studies presented herein made use of MPLA, a TLR4 agonist. To this end, Turbo preparations were created using the TLR1/2 heterodimerizing agonist Pam2CSK4 and the TLR 2/6 heterodimerizing agent Pam3CSK4, the structures of which are illustrated in FIG. 69 and the crystal structures of these homo and heterodimers are depicted in FIGs. 70, 71, and 72. FIGs 73 and 74 show a comparison of Turbo adjuvant formulation with various the TLR ligands. Wildtype (C57BL/6J) male and female mice were immunized i.m with 50 pl of phenol- extracted ViPS (2.5 micrograms) admixed with Turbo (containing 5 pg of MPLA, known TLR4 agonist); Turbo (containing 5 pg of Pam2CSK4, a known TLR2/6 agaonist); or Turbo (containing 5 pg of Pam CSK , a known TLR1/2 agaonist); ViPS-specific IgM and IgG isotype levels were then measured by ELISA. Overall, the TLR4 ligand MPLA generated the widest variety of antibody generation by stimulating significantly more IgM, IgG, IgG2b, IgG2c, and IgG3 than the other agonists, however PamsCSIGi induced a greater amount of IgGl, an equivalent amount of IgG and IgG2c to MPLA. Pai CSIGi generated an equivalent amount of IgG3 to MPLA but was much lower than MPLA in every other immunoglobulin type tested. The studies in FIG. 74 sought to compare PaimCSK-t to Pan CSB using the Typbar TCV ViPS and tetanus toxoid conjugate vaccine. Both formulations generated roughly similar levels of immunoglobulin that were not significantly different except IgG3, which Pam2CSK4 generated greater levels of.
Example 7: Turbo adjuvant enhances the immunogenicity of other types of vaccines
Having demonstrated the potential of Turbo as an adjuvant that enhances the immunogenicity of polysaccharide vaccines, its ability to enhance the response to other types of vaccines was then determined.
First, Turbo was combined with the H. influenzae PRP-conjugate vaccine Hiberix. Wildtype (CD1) (n = 8) male and female mice of 9 weeks were immunized i.m. with 50 pl of Hiberix® (1 micrograms with and without Turbo (containing 5 micrograms of MPLA). On day 14 and 28 of primary immunization mice were boosted and Polyribose Ribitol Phosphate (PRP)- specific IgG levels were measured by ELISA. See FIG. 62. Here, Turbo treated groups demonstrated significantly higher IgG production.
Likewise, Turbo adjuvant enhanced the effects of the Imovax Rabies viral vaccine. FIG. 63. Here, wildtype (C57BL6) (n = 8) male and female mice of 9 weeks were immunized i.m. with 50 pl of Imovax® (0.25 international Units) with and without Turbo (containing 5 micrograms of MPLA). Rabies virus glycoprotein (RVGP)-specific IgM and IgG levels were measured by ELISA. On day 35 of primary immunization mice were reimmunized as indicated, with the arrow. A subsequent study also examined Turbo’s adjuvant properties when combined with a haptenated model antigen, NP-CGG (FIG. 63). Here, groups of mice of various ages from 10 days to 90 weeks were used to approximate the use of Tubo/protein vaccines in infant, adult, and aged populations. The results demonstrated Turbo’s ability to stimulate significant production of IgG in all age groups.
Next, the performance of Turbo adjuvant was compared to alum and squalene, two adjuvants commonly used in human vaccines. Here, the adjuvants were combined with bacterial or synthetic polysaccharide, Protein conjugated polysaccharide vaccine or protein antigen (FIG. 64). The results of these studies found that Turbo stimulated significantly higher IgM and IgG against ViPS when compared to a squalene emulsion (FIG. 64A). When combined with the ViPS Tetanus Toxoid (TypBar TCV) vaccine, Turbo generated the highest amounts of ViPS- specific IgM and IgG, followed by squalene and alum (FIG. 64B). When the model antigen NP- CGG was used as an immunogen, alum generated higher IgGl titers, however Turbo generated higher IgM, IgG2b, IgG2c and IgG3 titers (FIG. 64C). Lastly, when NP-Ficoll was used as an immunogen, Turbo adjuvant generated greater amounts of all antibody types tested (FIG.64D).
The effect of the location of Turbo injection was then examined (Fig. 66). Male and female C57BL6 mice of 8-10 weeks of age were immunized i.m. in the right thigh of the hind limb with the model antigen NP-CGG, or protein conjugated- Vi PS vaccine (Typbar TCV) that were bedside mixed without or with Turbo. In a group of mice where the antigen was administered in the right thigh, and the adjuvant was injected in the left thigh (i.e., contralateral). NP-specific antibody and ViPS-specific antibody levels were then measured by ELISA. Results demonstrated that the adjuvant properties of Turbo require ipsilateral administration with the vaccine antigen.
Enumerated Embodiments
The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.
Embodiment 1 provides an immunogenic composition. In certain embodiments, the composition comprises one or more antigenic molecules between 0.01 mg/ml and 50.0 mg/ml. In certain embodiments, the composition comprises a surfactant at a concentration of at least 0.5% (w/w). In certain embodiments, the composition comprises an effective amount of a liposome adjuvant. In certain embodiments, the composition comprises a pharmaceutically acceptable carrier. In certain embodiments, the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pam 2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Pams CSK4) or an analogue or derivative thereof, and any combination thereof, and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
Embodiment 2 provides the immunogenic composition of embodiment 1, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
Embodiment 3 provides the immunogenic composition of embodiment 2, wherein the polysorbate 80 or an analogue or derivative thereof is between 0.1% and 10% (w/w).
Embodiment 4 provides the immunogenic composition of any one of embodiments 1-3, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a protein, a peptide, a peptide/polysaccharide conjugate, a protein/polysaccharide conjugate and any combination thereof.
Embodiment 5 provides the immunogenic composition of any one of embodiments 1-4, wherein the antigenic molecule is derived from a bacterium.
Embodiment 6 provides the immunogenic composition of any one of embodiments 1-4, wherein the antigen molecule is derived from a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite.
Embodiment 7 provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition. In certain embodiments, the composition comprises one or more antigenic molecules. In certain embodiments, the composition comprises a surfactant at a concentration of at least 0.5% (w/w). In certain embodiments, the composition comprises an effective amount of a liposome adjuvant. In certain embodiments, the composition comprises a pharmaceutically acceptable carrier. In certain embodiments, the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g. Pair CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g. Paim CSK4) or an analogue or derivative thereof, and any combination thereof, and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof.
Embodiment 8 provides the method of embodiment 7, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof.
Embodiment 9 provides the immunogenic composition of embodiment 8, wherein the polysorbate 80 is between 0.1% and 10% (w/w).
Embodiment 10 provides the method of any one of embodiments 7-9, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a peptide, and a peptide/polysaccharide conjugate.
Embodiment 11 provides the method of any one of embodiments 7-10, wherein the antigenic molecule is derived from a bacterium.
Embodiment 12 provides the method of any one of embodiments 7-11, wherein the antigenic molecule is derived from a virus.
Embodiment 13 provides the method of any one of embodiments 7-12, wherein the immune response is independent of T cells.
Embodiment 14 provides the method of any one of embodiments 7-12, wherein the immune response is dependent on T cells.
Embodiment 15 provides the method of any one of embodiments 7-14, wherein the immune response is B cell mediated.
Embodiment 16 provides the method of embodiment 15, wherein the B cells are stimulated by B cell receptor and toll-like receptor (TLR) signaling.
Embodiment 17 provides the method of any one of embodiments 15-16, wherein the B cells are Bib cells or Bib equivalent cells.
Embodiment 18 provides the method of any one of embodiments 7-16, wherein the immune response comprises the generation of antigen-specific antibodies.
Embodiment 19 provides the method of embodiment 18, wherein the antibodies comprise IgM antibodies, IgG antibodies, or a combination of IgM and IgG antibodies.
Embodiment 20 provides the method of any one of embodiments 7-18, wherein the immune response generates long-term immune memory.
Embodiment 21 provides the method of any one of embodiments 7-20, wherein the subject is human, primate, bovine, porcine, ovine, canine, feline or murine. Embodiment 22 provides a kit comprising the immunogenic composition of any one of embodiments 1-6.
Embodiment 23 provides a method of treating, ameliorating, and/or preventing a disease in subject in need thereof, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of embodiment 1-6.
Embodiment 24 provides the method of embodiment 23, wherein the disease is associated with the antigenic molecule.
Embodiment 25 provides the method of any one of embodiments 23-24, wherein the disease is a polysaccharide encapsulated bacterial infection.
Embodiment 26 provides the method of embodiment 25, wherein the polysaccharide encapsulated bacterial infection is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiritidis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptococcus, Pseudomonas aeruginosa, Klebsiella, Pasteurella, Brucella, Francisella, Helicobacter, Vibrio and Bacillus anthracis.
Embodiment 27 provides the method of any one of embodiments 23-26, wherein the bacterial infection is a non-polysaccharide encapsulated bacterial infection.
Embodiment 28 provides the method of embodiment 27, wherein the non-polysaccharide encapsulated bacterial infection is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Streptococcus, Chamydia, Escherichia coli, Meningococcal group B, Staphylococcus aureus, and Group A and B Streptococcus.
Other Embodiments
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is: An immunogenic composition comprising: one or more antigenic molecules between 0.01 Lig/ml and 50.0 pg/ml; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g., Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g., Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or a derivative thereof. The immunogenic composition of claim 1, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof. The immunogenic composition of claim 2, wherein the polysorbate 80 or an analogue or derivative thereof is between 0.1% and 10% (w/w). The immunogenic composition of any one of claims 1-3, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a protein, a peptide, a peptide/polysaccharide conjugate, a protein/polysaccharide conjugate and any combination thereof. The immunogenic composition of any one of claims 1-4, wherein the antigenic molecule is derived from a bacterium. The immunogenic composition of any one of claims 1-4, wherein the antigen molecule is derived from a pathogen selected from the group consisting of a virus, a fungus, a protozoan, and a multicellular parasite. A method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising: one or more antigenic molecules; a surfactant at a concentration of at least 0.5% (w/w); an effective amount of a liposome adjuvant; and a pharmaceutically acceptable carrier; wherein the liposome adjuvant comprises an effective amount of an adjuvant selected from the group consisting of Monophosphoryl Lipid A (MPLA) or an analogue or derivative thereof, Di-acyl lipopeptide (e.g., Pam2 CSK4) or an analogue or derivative thereof, Tri-acyl lipopeptide (e.g., Pam3 CSK4) or an analogue or derivative thereof, and any combination thereof; and l,2-dipalmitoyl-sn-glycero-3 phosphocholine (DPPC) or an analogue or derivative thereof. The method of claim 7, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) or an analogue or derivative thereof. The immunogenic composition of claim 8, wherein the polysorbate 80 is between 0.1% and 10% (w/w). The method of any one of claims 7-9, wherein the antigenic molecule is selected from the group consisting of a polysaccharide, a peptide, and a peptide/polysaccharide conjugate. The method of any one of claims 7-10, wherein the antigenic molecule is derived from a bacterium. The method of any one of claims 7-11 , wherein the antigenic molecule is derived from a virus. The method of any one of claims 7-12, wherein the immune response is independent of T cells. The method of any one of claims 7-12, wherein the immune response is dependent on T cells. The method of any one of claims 7-14, wherein the immune response is mediated by B cells. The method of claim 15, wherein the B cells are stimulated by B cell receptor and toll-like receptor (TLR) signaling. The method of any one of claims 15-16, wherein the B cells are Bib cells or Bib equivalent cells. The method of any one of claims 7-16, wherein the immune response comprises the generation of antigen-specific antibodies. The method of claim 18, wherein the antibodies comprise IgM antibodies, IgG antibodies, or a combination of IgM and IgG antibodies. The method of any one of claims 7-18, wherein the immune response generates long-term immune memory. The method of any one of claims 7-20, wherein the subject is human, primate, bovine, porcine, ovine, canine, feline or murine. A kit comprising the immunogenic composition of any one of claims 1-6. A method of treating, ameliorating, and/or preventing a disease in subject in need thereof, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of claim 1-6. The method of claim 23, wherein the disease is associated with the antigenic molecule. The method of any one of claims 23-24, wherein the disease is a polysaccharide encapsulated bacterial infection. The method of claim 25, wherein the polysaccharide encapsulated bacterial infection is selected from the group consisting of Salmonella typhi, Salmonella typhimurium, Salmonella entiritidis, Shigella, Salmonella paratyphi, Haemophilus influenzae, meningococcus, pneumococcus, Escherichia coli, group A or B Streptococcus, Pseudomonas aeruginosa, Klebsiella, Pasteurella, Brucella, Francisella, Helicobacter, Vibrio and Bacillus anthracis. The method of any one of claims 23-26, wherein the bacterial infection is a nonpolysaccharide encapsulated bacterial infection. The method of claim 27, wherein the non-polysaccharide encapsulated bacterial infection is selected from the group consisting of Bordetella pertussis, Clostridium tetani, Salmonella, Vibrio cholera, Pseudomonas aerugunosa, Corynebacterium diphtheriae, Gonococcus, Haemophilus, Streptococcus, Chamydia, Escherichia coli, Meningococcal group B, Staphylococcus aureus, and Group A and B Streptococcus.
PCT/US2023/082976 2022-12-08 2023-12-07 Immune adjuvants for polysaccharide vaccines WO2024124052A1 (en)

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US20150307545A1 (en) * 2005-02-07 2015-10-29 Lipotek Pty Ltd. Adjuvanting material
US20150335722A1 (en) * 2013-09-25 2015-11-26 Sequoia Sciences, Inc. Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections
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US20150307545A1 (en) * 2005-02-07 2015-10-29 Lipotek Pty Ltd. Adjuvanting material
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