WO2015073831A1 - Virtual conjugate particles - Google Patents

Virtual conjugate particles Download PDF

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Publication number
WO2015073831A1
WO2015073831A1 PCT/US2014/065716 US2014065716W WO2015073831A1 WO 2015073831 A1 WO2015073831 A1 WO 2015073831A1 US 2014065716 W US2014065716 W US 2014065716W WO 2015073831 A1 WO2015073831 A1 WO 2015073831A1
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Prior art keywords
protein
polysaccharide
antigenic
cross
pharmaceutical composition
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PCT/US2014/065716
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French (fr)
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Shyam M. Rele
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Liquidia Technologies, Inc.
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Publication of WO2015073831A1 publication Critical patent/WO2015073831A1/en

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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure is generally directed to pharmaceutical compositions that may be used in a vaccine. Methods for making the pharmaceutical compositions are also described.
  • Conjugate vaccines are typically created by covalently attaching an antigen to a carrier protein. Conjugation is often applied to bacterial polysaccharides for the prevention of bacterial diseases. Conjugation relies on specific chemistries to link the bacterial polysaccharide to the carrier protein. The conjugation process is complex and involves multiple steps. Both the protein and polysaccharide have to be manufactured, most likely including isolation and purification steps. In some cases, the polysaccharide needs to be sized. For an effective reaction, cierivatization may be required prior to chemical conjugation. After conjugation the composition must be purified to remove unreacfed materials. Some pathogens have multiple serotypes, for example pneumonia. A conjugation procedure that may be optimized with one serotype, may not work with another serotype of the same pathogen. And in some cases, the antigen is not amenable to the conjugation process at all.
  • the conjugation process is a chemical reaction
  • the relationship between antigen and carrier protein is a stoichiometric one.
  • U.S. Patent Application Publication 2010/0316666 to Hausdorfi et. ai the conjugation of serotypes of Streptococcus pneumonia with CRM is disclosed.
  • the table below details the conjugation ratios for the various serotypes.
  • a pharmaceutical composition where an antigen and a protein are present in appropnale spatial associalion which does not require the direct and difficult conjugation of protein to antigen would be highly desirable. Additionally, a pharmaceutical composition where antigen and protein can be combined in non-sioichiometric ratios would also be highly desirable.
  • FIG. 1 depicts a virtual conjugate particle of the invention.
  • FIG. 2 depicts a second embodiment of a virtual conjugate particle of the invention.
  • FIG. 3 depicts a third embodiment of a virtual conjugate particle of the invention.
  • FIG. 4 depicts a fourth embodiment of a virtual conjugate particle of the invention.
  • FIG. 5 depicts a one pot method of making a virtual conjugate particle of the invention.
  • FIG. 6 depicts a sequential method of making a virtual conjugate particle of the invention.
  • FIG. 7 depicts an admixture method of making a virtual conjugate particle of the invention.
  • FIG. 8 depicts the Anti-PnPs14 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #1 ).
  • FIG. 9 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
  • FIG. 10 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
  • FIG. 1 1 depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
  • FIG. 12 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
  • FIG. 13 depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
  • FIG. 14 depicts the Anti-PnPs14 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
  • FIG. 15 depicts the Anti-PLD IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
  • FIG. 16A depicts the Anti-PnPs1 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 16B depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 17A depicts the Anti-PnPs5 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 17B depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 18A depicts the Anti-PnPs14 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 18B depicts the Anti-PnPs14 IgG Reciprocal Titer and the Anti-PnPs14 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
  • FIG. 19 depicts the Anti-Vi IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Typhoid Mouse Study).
  • FIG. 20 depicts the Anti-Vi IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Typhoid Mouse Study).
  • FIG. 21 depicts ELISpot and Anti-Flu HA IgG ELISA Titer for virtual conjugate particles produced according to the invention tested in a mouse model (RNA Mouse Study).
  • FIG. 22 depicts ELISpot and Anti-RSV-F IgG ELISA Titer for virtual conjugate particles produced according to the invention tested in a mouse model (RNA Mouse Study).
  • FIG. 23A depicts Anti-PnPs 6A titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #4).
  • FIG. 23B depicts Anti-PnPs 19A titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #4).
  • ADH adipic dihydrazide
  • BSA bovine serum albumin
  • CRM 197 or CRM 197 genetically detoxified mutant of diphtheria toxin cRPMI: complete Roswell Park Memorial Institute medium CWPS/22F: cell wall polysaccharide/22F DC-Chol: cholesteryl 3 -N-(di-methyl-amino-ethyl)-carbamate, DC-cholesterol
  • EDTA ethylenediaminetetraacetic acid
  • FBS fetal bovine serum
  • HA-RNA self-replicating RNA encoding for HA (hemagglutinin) RNA
  • immunoglobulin G immunoglobulin G
  • OPK opsonophagocytic killing assay
  • OVA ovalbumin
  • PBS phosphate buffered saline
  • PLD genetically detoxified mutant of pneumolysin toxin
  • PLGA poly(lactide-co-glycolide) polymer
  • PMA phorbol 12-myristate 13-acetate
  • PnP or PnPs purified pneumococcal polysaccharide
  • PNPP p-nitrophenyl phosphate
  • RSV-F RNA self-replicating RNA encoding for RSV-F (respiratory syncytial virus protein F) RNA
  • virtual conjugate particles which contain multiple-layered particles typically comprising a core particle.
  • the virtual conjugate particle approach described herein provides an adaptable p!atform for producing medicines for the treatment and prevention of disease.
  • the pharmaceutical composition generally includes an active agent associated with the particle and a highly charged protein through reacting a cross-linker with the particle, active agent, and charged protein.
  • the pharmaceutical composition may also contain a carrier protein, antigen substance, antigenic protein, adjuvant(s) such as alum (i.e. AlydrogeL Adjuphos), and/or therapeutics.
  • the pharmaceutical composition of the present invention comprises: i) a polymeric core particle,
  • an active agent such as, for example, an antigen, drug, adjuvant, or biologic, and in some embodiments a polysaccharide or a nucleic acid, iii) a charged biomoiecuie such as a protamine protein,
  • Virtual conjugate particles of the invention are produced by association of an active agent (for example, an antigenic polysaccharide or a nucleic acid) with a charged biomoiecuie (for example, a protamine protein).
  • the virtual conjugate particles typically contain a polymeric core particle that provides a surface for the association of the agent with the charged biomoiecuie.
  • a cross-linker may be used to covalently cross-link one or more particle components.
  • the cross-linker may link the charged biomoiecuie (e.g. prolamine) to itself, allowing the particle to physically entangle the agent (e.g., a polysaccharide).
  • the agent e.g., a polysaccharide.
  • physical entanglement does not include a covaleni interaction.
  • physically entanglement may not include an electrostatic interaction.
  • the cross-linker may link the charged biomoiecuie to Ihe agent.
  • the cross-linker may covalently link a virtual conjugate particle component to a carrier protein. Cross-linking one or more components to each other may provide for enhanced stability of the virtual conjugate particle.
  • the virtual conjugate particles are prepared so as to prevent cross- linking of certain components.
  • the agent e.g. polysaccharide or protein antigen
  • carrier protein are not linked covalently to each other.
  • the virtual conjugate particles are especially useful where the active agent is an antigen.
  • Virtual conjugation places antigens in appropriate spatial orientation with protein without requiring covended bonding found in conjugation-based approaches.
  • the virtual conjugation approach process is not limited to having a required sloichiometry between two molecules. Virtual conjugation therefore provides advantages over old approaches in the art.
  • virtual conjugation approaches disclosed herein are less sensitive to polysaccharide sizing limitations. Conjugation to a carrier can require that an antigen, for example a polysaccharide, is appropriately sized so as to achieve efficient conjugation to a carrier moiety.
  • the number of saccharide repeats in a polysaccharide may not be optimal for inducing an immune response, in contrast, the virtual conjugation approaches herein facilitate enhanced immune responses by providing the option of administering particles containing larger polysaccharides.
  • the surface can be used to adsorb the antigen or provide a scaffold.
  • the polymeric core particle surface area is high and can be modulated by the size and/or configuration of the polymeric core particle. Multiple copies of an antigen can be associated with the surface of a single core particle. The number of copies is not bounded by stoicbiometry.
  • polymeric core particle simplifies manufacturing and provides a versatile virtual conjugate particle system.
  • the polymeric core particle is surface decorated or surface coated with antigen(s) and/or protein ⁇ s) of interest.
  • a cross-linker associates the materials with the surface of the polymeric core particle in a non-stoichiometric ratio.
  • stock solutions allows layering of antigen(s) and/or protein(s) of interest. No modification, such as derealization, is necessary for either the antigen or the protein prior to addition. Selection of an antigen/prolein combination is not dictated by conjugation chemistries.
  • An antigen can be combined with a protein of choice at a ratio of choice.
  • Microparticles and/or nanoparticies are known in the art. Various types include those produced from microemu!sions including oil in water (Q/W), water in oil (W/O), and water in oil in water (W/O/W) microparticles.
  • beads/microbeacls/microspheres such as those constructed of various polymers such as polystyrene, functionaiized polystyrene, polymethylmethacrylate, poly( -caproiactone), polylactide, or poiyiiactide-co-glycolide) polymer to name a few.
  • Microparticles and/or nanoparticies fabricated through PRINT ⁇ Technology offers microparticles and/or nanoparticies with control over their chemical composition, particle size, particle shape, surface functionality, and other physical and chemical characteristics.
  • PRINT ⁇ Technology Liquidia Technologies, inc., or svilie, North Carolina
  • Use of this technology in the present invention allows for the co-packaging of antigen(s) and/or carrier proteini c s ⁇ to stimulate an immune response without the use of conjugation.
  • a core particle provides a large surface for the association of the active agent, charged biomoiecule, and carrier protein.
  • the table below details the molecular weights for some exemplary core particles, antigenic proteins, and polysaccharides.
  • the weight of the core particle is several orders of magnitude greater than that of any of the active agents and/or any of the carrier proteins/antigenie proteins.
  • the surface of the very large core particle provides a very large area for association of numerous molecules of active agent, carrier protein, and/or antigenic protein. The association is not bound by a specific stoichiometric value such as that found in the conjugation process.
  • Material Approximate Molecular Weight, kDa Materials Approximate Molecular Weight, kDa
  • Suitable materials for use in the invention to produce core partic!es comprise a biocompatible polymer.
  • the polymer is selected from the group consisting of a polyester, a polyanhydride, a polyamide, a phosphorous-based polymer, a poiy(cyanoacrylate), a polyorthoester, a polyurethane, a polyorthoester, a poiyether, a carbohydrate, a polypeptide, a hydroxypropyicelluiose, a poly(ethylene glycol), a wax, a hydrogel, a phosphatidylcholine, a poiydihydropyran, a polyacetal, a biodegradable polymer, and combinations thereof, in some embodiments, the polyester is selected from the group consisting of po!y!acUc acid, po!y!acUde, po!yglyco!ic acid, poly(hydroxybulyrate), polyis- caprolactone
  • the polyanhydride is selected from the group consisting of poiyisebacic acid), poly(adipic acid), and po yiterephtbalic acid), in some embodiments, the po!yamide is selected from the group consisting of po!y(Smino carbonates) and polyaminoacids.
  • the phosphorous-based poiymer is selected from the group consisting of polyphosphate, a polyphosphonate, and a polyphosphazene.
  • the polymer is po!ylactide, polyglycolic acid, or poly(iactide ⁇ co ⁇ giycolide) polymer. More preferably the polymer is poiyiactide or poly(iactide-co-glycolide) polymer. Most preferably the polymer is poiyilactide-co-glycolide) polymer.
  • the particle may also be comprised of a protein.
  • Such particles and methods to make such particles are described in WO2008/106503 which is incorporated herein by reference in its entirety.
  • the virtual conjugate particles of the present invention comprise one or more active agents useful for exerting a biological effect on a subject.
  • active agents include, for example, drugs, small molecules, proteins, nucleic acids, oligonucleotides, natural oligosaccharides, synthetic oligosaccharides, polysaccharides, adjuvants, antibodies, antibody fragments, and biologies.
  • the active agent used in the present invention is preferably a polysaccharide or a nucleic acid.
  • Suitable polysaccharides for use in the invention include bacterial polysaccharides as well as carbohydrates of cancer cells or other non-self or target cell carbohydrates.
  • the polysaccharides are the pneumococcal polysaccharides PnPs PnPsS, PnPs 6A and PnPs14.
  • the polysaccharides are from Pneumovax, Meningococcal, typhoid, cell surface glycolipids, glycoproteins, Haemophilus influenza Type b (Hib), RSV-F, staph, chlamydia, meningococcal type B, (Mening B), C. Difficile, Pseudomonas, Group A & B strep, enterotoxigenic Escherichia coii (ETEC), tuberculosis (IB), malaria toxin, Candida albicans, Shigella, Salmonella typhi, Salmonella paratyphi A, B, and C, C. botulinum, plague, Burkholcieria, and the like.
  • Exemplary PnPs polysaccharides include, but are not limited to, PnPsl , PnPs2, PnPs3, PnPs4, PnPsS, PnPs 6A, PnPsob, PnPs7F, PnPsS, PnPs9N, PnPs9V, PnPsl OA, PnPs1 1A, PnPs12F, PnPs14, PnPs15B, PnPs17F, PnPs18C, PnPs19F, PnPs19A, PnPs20, PnPs22F, PnPs23F, and PnPs33F of Streptococcus pneumoniae.
  • the amount of polysaccharide used in the fabrication of ihe virtual conjugate particle may be described with respect to the amount of the core particle.
  • the amount of polysaccharide may be described with respect to the core particle: polysaccharide weight ratio.
  • the ratio of core particle to polysaccharide may be about 1 :0.001 (w:w), about 1 :0.002 (w:w), about 1 :0.003 (w:w), about 1 :0.004 (w:w), about 1 :0.005 (w:w), about 1 :0.01 (w:w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), or about 1 :0.05 (w:w).
  • the polysaccharide used in the fabrication may be present in a range of about 1 :0.001 (w/w) to about 1 :0.05 (w/w).
  • the polysaccharide is in a range of about 1 :0.01 (w:w) to about 1 :0.05 (w:w) (w/w).
  • the range is about 1 :0.01 (w:w) to about 1 :0.03 (w/w).
  • the amount of polysaccharide used in the fabrication of the virtual conjugate particle may also be expressed as a percentage of the core particle amount.
  • the polysaccharide may be present at about 0.1 % (w/w) of the core particle, about 0.2% (w/w) of the core particle, about 0.3% (w/w) of the core particle, about 0.4% (w/w) of the core particle, about 0.5% (w/w) of the core particle, about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, or about 5% (w/w) of the core particle.
  • the polysaccharide used in the fabrication of the virtual conjugate particle may be present in a range of about 0.1 % to about 5% (w/w).
  • the polysaccharide is in a range of aboul 0.1 % to about 5% (w/w).
  • the range is aboul 1 % to about 3% (w/w).
  • bui are ⁇ limited to: pneumoiysoid protein (PLD), pneumococoai surface protein A (PspA), choline binding protein A (Cbp.A), PcsB- ⁇ proiein required for cell wall separation of group B streptococcus) and serine/threonine protein kinase (StkP) (inierceli), pneumococcal choline- binding protein A (PcpA), pneumococcal surface adhesion A (a substrate-binding lipoprotein of a manganese ABC transporter) (PsaA), serine/threonine protein kinase (StkP), pneumococcal hislidine triad proteins (PhtA B/D/E), and protein required for cell separation of group B streptococcus (PcsB).
  • PLD pneumoiysoid protein
  • PspA pneumococoai surface protein A
  • Cbp.A
  • Suitable nucleic acids for use in the invention include RNA, DNA, siRNA, shRNA, dsRNA, ssRNA, mi RNA, mRNA, rRNA, 1RNA, snR A, ssDNA, dsDNA, plasmid DNA, self- replication RNA, and antisense RNA.
  • the particles may contain a layer of charged biomolecuies that associate with the core particle structure and one or more particle components, including polysaccharides, linkers, and carrier proteins, by virtue of the electrostatic charge of the charged biomoiecuie.
  • an interaction may also comprise a covalent link, typically introduced using a cross-linker after the initial electrostatic-based interaction.
  • the charged biomoiecuie may be a caiionic biomoiecuie.
  • the caiionic biomoiecuie may be selected from poiyeiecirolytes, charged polyaminoacids, charged polysaccharides, charged proteinaceous compounds, and charged peptides.
  • Examples of positively charged biomolecuies include, but are not limited to, polyamino acids such as poiyiysine, polyhistidine, poiyornithine, polyciirulline, polyhydroxylysine, polyarginine, poiyhomoarginine, poiyaminotyrosine, histones, and protamines/protamine salts.
  • polyamino acids such as poiyiysine, polyhistidine, poiyornithine, polyciirulline, polyhydroxylysine, polyarginine, poiyhomoarginine, poiyaminotyrosine, histones, and protamines/protamine salts.
  • Suitable positively charged biomolecuies include, but are not limited to, polydiaminobulyric acid, poiyetbyieneimine, polypropyleneimine, polyamino(meth)acryl3te, polyaminosiyrene, poiyaminoethylene, poly(aminoetbyi)ethylene, polyaminoethylstyrene, diethyl amino ethyl cellulose, poiy-imino tyrosine, cholestyramine-resin, poly-imino acid, 1 ,5-dimethyi-1 ,5 ⁇ diazaundecametbylene poiymeihobromide (hexadimethrine bromide), chitosan, poiy(amidoamine) dendrimers, and combinations thereof
  • Cationic biomoiecules of the present disclosure typically comprise one or more positively charged monomers.
  • positively charged monomers include, but are not limited to, lysine, histidine, ornithine, hydroxyiysine, arginine, homoarginine, aminotyrosine, diaminobutyric acid, ethyieneimine, propyienimine, amino(meth ⁇ acryiaie, aminostyrene, aminoethylene, aminoethylethyiene, aminoethylstyrene, citruiline, diethyl amino ethyl glucose, imino tyrosine, (vinyibenzyi)trirnethyiammonium salts, imino acids, quaternary aikyi ammonium salts, amidoamines, glucosamine, and mixtures and derivatives thereof.
  • the charged biomoiecule may be a synthetic heteropolymer polypeptide containing only positively charged amino acids.
  • the charged biomoiecule may be a heteropolymer polypeptide of at least two of arginine, lysine, and histidine in a repeating unit, in some aspects, the repeating units are dimers or 1 Brimmers; for example, Arg ⁇ Lys dimers or Arg-Lys-His 16rimmers.
  • the polypeptides may contain repeated combinations of dimers and 18rimmers.
  • the arginine, lysine, and histidine residues rnay be arranged in any order.
  • the heteropolymer polypeptide may contain non-positively charged amino acids. Typically, however, the heteropolymer polypeptide mostly contains positively- charged amino acids; for example, the polypeptide contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% positively-charged amino acids. Such polypeptides are readily synthesized by recombinant techniques.
  • the cationic biomoiecule is a protamine protein or a protamine protein salt. More preferably the cationic biomoiecule is a protamine salt.
  • protamine salts include protamine sulfate, protamine chloride, protamine acetate, protamine propionate, protamine lactate, protamine formate, protamine nitrate, and protamine acetate.
  • Most preferab y the cationic biomo!ecuie is protamine sulfate.
  • the amount of caiionic biomoiecuie used to fabricate the virtual conjugate particle may be described with respect to the amount of the core particle, in particular, the amount of cationic biomo!ecu!e may be described with respect to the core particle: cationic biomo!ecu!e weight ratio.
  • the ratio of core particle to cationic biomoiecuie may be about 1 :0.01 (w:w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), about 1 :0.05 (w:w), about 1 :0.06 (w:w), about 1 :0.07 (w:w), about 1 :0.08 (w:w), about 1 :0.09 (w:w), or about 1 :0.1 (w:w).
  • the polysaccharide may be present in a range of about 1 :0.01 (w/w) to about 1 :0.1 (w/w).
  • the polysaccharide is in a range of about 1 :0.03 (w:w) to about 1 :0.08 (w:w) (w/w). Often, the range is about 1 :0.04 (w:w) to about 1 :0.08 (w/w).
  • the amount of cationic biomoiecuie used to fabricate the virtual conjugate particle may also be expressed as a percentage of the core particle amount.
  • the cationic biomoiecuie may be present at about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, about 5% (w/w) of the core particle, about 6% (w/w) of the core particle, about 7% (w/w) of the core particle, about 8% (w/w) of the core particle, about 9% (w/w) of the core particle, or about 10% (w/w) of the core particle.
  • the cationic biomoiecuie may be present in a range of about 1 % to about 10% (w/w). Typically, the cationic biomoiecuie is in a range of about 3% to about 8% (w/w). Often, the range is about 4% to about 6% (w/w). Methods of producing particles to achieve these ratios are described in Example 9.
  • the particles may be modified by chemically cross-linking the charged protein, entangling the antigenic substance near the surface of the particle.
  • the proteins can be chemically cross-linked either through the nitrogen functionality of N-ierminus or lysine residues or the thiol functionality of cysteine residues using an appropriate difunctional cross-linker.
  • proteins can be chemically cross-linked through carboxyiates to primary amines.
  • Suitable cross-linkers for use in the invention include, but are not limited to 1-ethyi-3-(3- dimethylaminopropyi) carbodiimide (EDC), giutaraidehyde (GA), bismaleimidohexane (BMH), dimethyl suberirnidate (DMS), dimethyl pime!imidaie (D P), 4-(4,6 ⁇ dimethoxy-1 ,3 l 5-trianzi-2 ⁇ yl) ⁇ 4-methylmorphoiiniurn chloride, 1-cyano-4-dimetbyiaminopyndinium tetrafiuoroborate (CDAP), N-hydroxysuccinimide esters (NHS), dithiobis(succinimidyi propionate(DTSP), 4-(4,6 ⁇ dimethyl- 1 ,3,5-triazin-2-yi) (DMTMM) Des Martin Periodinane, Tempo/ aGiG 4 , 2-iminoihioione,
  • the N-hydroxysuccinimide ester is selected from the group consisting of dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), succinimidyl 3 ⁇ (2 ⁇ pyhdydithio) propionate (SPDP), succinimydyl 4 ⁇ [N- maieimidomethyl] cyciohexane-1-carboxyiate (SMCC), sulfosuccinimidyi-4-[N-rnaleimidomethyi] cyclehexane-1-carboxylate (suifo-S CC), and sulfosuccinimidyl 8-(3'-[2- pyridydithiojpropionamido) hexanoate (suifo-LC-SPDP).
  • DSP dithiobis(succinimidylpropionate)
  • the cross-linker is 1-ethyl-3-(3-dimethyiarninopropyi) carbodiimide (EDC), giutaraidehyde (GA), aclipic dihydrazide (ADH), or bismaleimidohexane (BMH). More preferably the cross-linker is 1-ethyl ⁇ 3 ⁇ (3-dimethyiaminopropyi) carbodiimide (EDC) or giutaraidehyde (GA). Most preferably the cross-linker is giutaraidehyde (GA).
  • the antigenic polysaccharide is PnPsI , PnPs 6A, or typhoid
  • the cross-linker is EDC.
  • the antigenic polysaccharide is PnPsI , PnPs5, or PnPs14
  • the cross-linker is glutaraidehyde (GA).
  • the antigenic polysaccharide is PnPs 6A and the cross-linker is ADH.
  • the pharmaceutical composition particle of the present invention may also include a carrier protein.
  • Suitable carrier proteins for use in the invention include, but are ⁇ limited to CRM tetanus toxoid, diphtheria toxoid, hemagglutinin, meningococcal surface proteins, pseudomonas aeruginosa endotoxin A (r-EPA), T-cei! protein antigens, ovalbumin, and capsular proleins.
  • CR 97 is an example of a non-toxin, mutant protein derived from diphtheria ioxin (DT) and that has long been recognized to be a non-toxic protein and is commonly used as a carrier for conjugate vaccines.
  • this protein has been utilized for various purposes, including as an inhibitor of beparin-binding EGF-like growth factor (HB-EGF) and as an immunological adjuvant for vaccination.
  • HB-EGF beparin-binding EGF-like growth factor
  • suitable carrier proteins include, but are ⁇ limited ⁇ S.
  • Aureus aggiuUnin-like sequence (Ais), including Ais3, which promotes adhesion to the endotheiiial cells ⁇ also for an antigen for Candida) (Novadigm), dumpling Factor A (ClfA) (Wyetb/Pfizer), clumping factor 8 (CifB), iron surface determinant B (isdB), Can-FnBP fusion protein, Poly-M-acetyl glucosamine; meningitis B; GNA 1870, also named factor H- binding protein (fHbp) or rLP-2086 (Novartis); Group B Strep: Protein Antigens c, R and X.ln some cases, an active agent can also act as a carrier protein.
  • an active agent can also act as a carrier protein.
  • Examples include, but are not limited to: pneumolysoid protein (PLD), pneumococcal surface protein A. (PspA), choline binding protein A (CbpA), Pcs8- ⁇ protein required for cell wail separation of group B streptococcus) and serine/threonine protein kinase (StkP) (Intercell), pneumococcal choline-binding protein A (Pc-pA), pneumococcal surface adhesion A ⁇ a substrate-binding lipoprotein of a manganese ABC transporter) (PsaA), serine/threonine protein kinase (StkP), pneumococcal bistidine triad proteins (PbtA/B/D/E), and protein required for ceil separation of group B streptococcus (PcsB), [0040]
  • the carrier protein is pneumolysold protein (PLD), CRM-
  • the amount of carrier protein used to fabricate the virtual conjugate particle may be described with respect to the amount of the core particle.
  • the amount of carrier protein may be described with respect to the core particle: carrier protein weight ratio.
  • the ratio of core particle to carrier protein may be about 1 :0.01 ( :w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), about 1 :0.05 (w:w), about 1 :0.06 (w:w), about 1 :0,07 (w:w), about 1 :0.08 (w:w), about 1 :0.09 (w:w), or about 1 :0.1 (w:w).
  • the carrier protein may be present in a range of about 1 :0.01 (w w) to about 1 :0.1 (w/w).
  • the polysaccharide is in a range of about 1 :0.03 (w:w) to about 1 :0.08 (w:w) (w/w).
  • the range is about 1 :0.04 (w:w) to about 1 :0.08 (w/w).
  • the amount of carrier protein used to fabricate the virtual conjugate particle may also be expressed as a percentage of the core particle amount.
  • the carrier protein may be present at about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, about 5% (w/w) of the core particle, about 6% (w/w) of the core particle, about 7% (w/w) of the core particle, about 8% (w/w) of the core particle, about 9% (w/w) of the core particle, or about 10% (w/w) of the core particle.
  • the carrier protein may be present in a range of about 1 % to about 10% (w/w). Typically, the carrier protein is in a range of about 3% to about 8% (w/w). Often, the range is about 4% to about 6% (w/w). Methods of producing particles to achieve these ratios are described in Example 9
  • the amount of polysaccharide and carrier protein in the virtual conjugate particles may also be described with respect to the ratio between these two components.
  • the ratio of polysaccharide to carrier protein may be in a range between about 0.001 and about 0.5, between about 0.01 and about 0.5, between about 0.01 and about 0.4, and between about 0,02 and about 0.4. In preferred aspects, the ratio is between about 0.02 and about 0.35.
  • the particle can include other agents, excipients or stabilizers.
  • negatively charged components include, but are not limited to bile salts of bile acids consisting of glycocholic acid, cholic acid, chenodeoxycholic acid, taurochoiic acid, giycochenodeoxychoiic acid, taurochenodeoxychoiic acid, iitochoiic acid, ursodeoxycholic acid, dehydrocholic acid and others: phospholipids including lecithin (egg yolk) based phospholipids which include the following phosphatidylcholines: paimitoyioleaylphosphaticiylchoifne, paimitoyilinoieoyiphosphatidyichoiine, stearoyilinoieoyiphosphatidyicholine stearoyioie
  • phospholipids including L-o dimyristoylpbospbatidyicholine (DMPC), dioieoylphosphatidyicholine (DOPC), distearoylphosphatidylchoiine (DSPC), hycirogenateci soy phosphatidylcholine (HSPC), and other related compounds.
  • Negatively charged surfactants or emulsifiers are also suitable as additives, for example, sodium cholesieryi sulfate and the like.
  • the positive zeta potential of the core particles can be altered by adding positively charged components.
  • Such positively charged components incksde, but are not limited to cationic lipids such as 1 ,2-dioieoyl- 3-trimethy!ammoniumpropane chloride (DOTAP), and 3 ⁇ -[ ⁇ -( ⁇ N'-dirnethyiaminoetbane)- carbamoyl] cholesterol hydrochloride (DC-Ghol).
  • DOTAP 1 ,2-dioieoyl- 3-trimethy!ammoniumpropane chloride
  • DC-Ghol 3 ⁇ -[ ⁇ -( ⁇ N'-dirnethyiaminoetbane)- carbamoyl] cholesterol hydrochloride
  • the conjugate particles have also found use for delivery of a pharmaceutical composition, such as a vaccine, that may be administered via various routes including orally, derrnally, inhalation, intravenously, intramuscularly, subcutaneousiy, intra-artehaily, intraperitoneally, intranasally, inlrapulmonary, intraoculariy, intravasculahy, intralhecaiiy, or intratracheal!'/.
  • a pharmaceutical composition such as a vaccine
  • routes including orally, derrnally, inhalation, intravenously, intramuscularly, subcutaneousiy, intra-artehaily, intraperitoneally, intranasally, inlrapulmonary, intraoculariy, intravasculahy, intralhecaiiy, or intratracheal!'/.
  • FIG. 1 depicts one embodiment of a pharmaceutical composition particle 100 of the invention.
  • the pharmaceutical composition particle 100 comprises a base particle 101 with a cationic biomoiecule 102 associated with the surface of the core particle.
  • a polysaccharide 103 and a carrier protein 104 Associated with the cationic biomoiecule 102 are a polysaccharide 103 and a carrier protein 104.
  • a cross-linker 105 is associated with the polysaccharide 103, carrier protein 104, and cationic biomoiecule 102.
  • the core particle 101 is poly(iactide-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide 103 is pneumococcal polysaccharide PnPs 5, carrier protein 104 is PLD, and cross-linker 105 is giutaraidehyde, in another embodiment, the core particle 101 is poly(lactide-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide is pneumococcal polysaccharide PnPs 14, carrier protein 104 is PLD, and cross-linker 105 is giutaraidehyde.
  • the core particle 101 is poly(!actide-co-giycolide) polymer
  • cationic biomoiecule 102 is protamine sulfate
  • polysaccharide is pneumococcal polysaccharide PnPs 1
  • carrier protein 104 is PLD
  • cross- linker 105 is 1 -ethyl-3-[3-dimethylaminopropyi]carbodiimide hydrochloride (EDC).
  • the core particle 101 is poly(lacticie-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide 103 is pneumococcal polysaccharide PnPs 14, carrier protein 104 is ovalbumin (OVA), and cross-linker 105 is glutaraldehyde.
  • the core particle 101 is poly(lacUde-co-giycolide) polymer, cationic biomolecule
  • polysaccharide 103 is pneumococcal polysaccharide PnPs 14
  • carrier protein 104 is CR ⁇ T
  • cross-linker 105 is glutaraldehyde.
  • FIG. 2 depicts a second embodiment of a virtual conjugate particle 200 of the invention.
  • the virtual conjugate particle 200 comprises a core particle 101 with a carrier protein 104 and polysaccharide 103 associated with the surface of the particle.
  • a cross-linker 105 is associated with the polysaccharide 103 and carrier protein 104.
  • the core particle 101 is poly(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide 103 is pneumococcal polysaccharide PnPs 5, carrier protein 104 is PLD, and cross-linker 105 is glutaraldehyde.
  • the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide
  • the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol
  • polysaccharide 103 is pneumococcal polysaccharide PnPs 1
  • carrier protein 104 is PLD
  • cross-linker 105 is bismaleimidohexane (B H).
  • the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol
  • polysaccharide 103 is Vi polysaccharide
  • carrier protein 104 is CRM 5g -
  • cross-linker is glutaraldehyde.
  • FIG. 3 depicts a third embodiment of a virtual conjugate particle 300 of the invention.
  • the virtual conjugate particle 300 comprises a core particle 101 with a cationic biomolecule 102 associated with the surface of the core particle.
  • a nucleic acid 106 Associated with the cationic biomolecule 102 is a nucleic acid 106.
  • the core particle 101 is poly(iactide-co-giycolide) polymer, cationic biomolecule 102 is protamine sulfate, and nucleic acid 106 is influenza HA R A.
  • ihe core particle 101 is poly(iactide ⁇ co-giycolide) polymer, cationic biomolecu!e 102 is protamine sulfate, arid nucleic acid 108 is RSV-F RNA,
  • FIG. 4 depicts a fourth embodiment of a virtual conjugate particle 400 of the invention.
  • the virtual conjugate particle 400 comprises a core particle 101 with a nucleic acid 106 and a cationic biomolecule 102 associated with the surface of Ihe particle.
  • the core particle 101 is poly ⁇ lacticle-co-glycolide) polymer blended with DC-cholesterol, nucleic acid is influenza HA RNA, and cationic biomolecule 102 is protamine sulfate.
  • One aspect of the present invention is the surface coating of the core particles with a charged species. Additional components can then be adsorbed to the surface of the core particles after application of the surface coating. These components can be charged or neutral moieties including but not limited to proteins (OVA, CRM, TT, PLD), polysaccharides (Vi PnPs, pneumococcal PnPs strains, synthetic PnPs), and nucleic acids.
  • core particles with surface adsorbed components can be treated with a stoichiometric excess in relation to the amount of protein being added of a cross-linking agent,
  • FIG. 5 depicts a "one pot" process for producing the virtual conjugate particle 100 of the invention.
  • a plurality of core particles 101 are suspended in water or buffer solution.
  • a cationic biomolecule 102 is added to the suspension and the combination is agitated with rocking at room temperature.
  • polysaccharide 103 is added to the suspension and the combination is agitated with rocking at room temperature.
  • carrier protein for example approximately ten minutes
  • the overall volume of the reaction is maintained at 1 mL 2 mg of poly (lactic acid-co-glycoiic acid) core particles 101 are suspended in water or a buffer solution.
  • 44.94 pg of pneumococcal polysaccharide PnPsl (in solution), a polysaccharide 103 is added to the suspension without removing the previous solution.
  • FIG. 6 depicts a second process for producing the virtual conjugate particle 100 of the invention.
  • a plurality of core particles 101 are suspended in water or buffer solution.
  • the cationic biomoiecule 102 is added to the suspension and incubated with the core particles 101 for a period of time, for example overnight, with rocking.
  • the resulting coated core particles are centrifuged, washed and re-suspended in water or buffer solution.
  • Polysaccharide 103, carrier protein 104, and cross-linker 105 are added to the suspension which is then incubated for a period of time, for example approximately one hour, at room temperature with rocking.
  • the cross-linker 105 is neutralized, if applicable, and the resulting virtual conjugate particle 100 is Ihen centrifuged, washed and recovered from the suspension through centrifugation.
  • a plurality of poly (lactic acid-co-glycoiic acid) core particles 101 are suspended in water or a buffer solution.
  • a 5% protamine sulfate solution, a cationic biomolecuie 102, are added and the suspension is rocked at room temperature overnight.
  • the unbound protamine is then removed using centrifugation and water washing.
  • Pneumococcal polysaccharide PnPsS or PnPs 14 2% solution
  • carrier protein PLD 4% solution
  • glutaraldehyde glutaraldehyde (1 % solution) are then added and the suspension is rocked for approximately one hour at room temperature.
  • 35m sodium cyanoborohydride (NaCNBH 3 ) is added to quench the reaction.
  • the pharmaceutical composition is recovered through centrifugation and washing of the particles.
  • FIG. 7 depicts a third process for producing the virtual conjugate particle 100 of the invention
  • a cationic biomolecuie 102 and carrier protein 104 are combined in a solution and incubated for a period of time, for example approximately one hour.
  • This solution is then added to a suspension of core particles 101 and mixed for a period of time, for example approximately one hour, at room temperature with agitation.
  • the resulting particles are centrifuged, washed and re-suspended in water or buffer solution.
  • Polysaccharide 103 and cross-linker 105 are added to the suspension and the mixture is incubated for a period of time, for example overnight, at room temperature with rocking.
  • the cross-linker is neutralized, if applicable, and the resulting virtual conjugate particles are centrifuged, washed and recovered from the suspension through centrifugation,
  • a 5% protamine sulfate solution and a 4% PLD carrier protein solution are combined in a 0.5 mL tube and allowed to incubate at room temperature for one hour.
  • the protamine/protein mixture are added to a plurality of poly (lactic acid-co-giycolic acid) core particles.
  • the suspension is rocked in the tube at room temperature for one hour. Unbound protamine and/or protein are removed using centrifugation and water washing.
  • Pneumococcal polysaccharide PnPsl 2% solution
  • 1 ⁇ ethyl-3-[3 ⁇ dimeihylaminoprapyl carbodiirnide hydrochloride (EDC) are added.
  • the pharmaceutical composition is recovered through centrifugation and washing of the particles.
  • compositions of the present invention may be used for both prophylactic and therapeutic purposes.
  • the particles and compositions containing the particies may be administered to any subject in need.
  • the subject is a mammalian subject.
  • the mammalian subject is a human, in other aspects, the particle and composition may be used for veterinary purposes and administered to an animal may it be a cat, dog or other animal
  • the invention provides for the use of a vaccine for the treatment of human patients.
  • the invention further provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient, in particular, the invention provides a method of treating viral, bacterial, or parasitic infections or cancer which comprises administering an effective amount of a drug delivery composition of the present invention to a patient.
  • An appropriate vehicle for administration of the drug delivery composition is a solution or suspension containing the particles.
  • the particles can also be part of a lyophilized composition prior to administration.
  • the lyophilized composition can be reconsititued into a solution or suspension prior to administration.
  • the particles may be delivered through various routes including subcutaneously, intravenously, intraderrnaliy, or intramuscularly in a solution or suspension.
  • the pharmaceutical composition may contain the target amount of antigenic material in an aqueous solution of mannitoi and polyvinyl alcohol, containing for example (wt%) 5% mannilol and 0.1 % polyvinyl alcohol,
  • the virtual conjugate particles may contain a single antigen or multiple antigens of interest.
  • Virtual conjugate particles may be blended to produce a pharmaceutical composition with a multitude of antigens.
  • three monovalent virtual conjugate particle compositions can be blended to form a pharmaceutical composition, in this example, three separate monovalent particle suspensions of known particle concentrations, in mg/mL, are combined into a trivalent particle suspension by transferring a calculated volume of each particle suspension into one vial.
  • the calculated volume contains a known mass of particles based on the particle concentration of each suspension.
  • the particle suspension can be from a freshly made batch of virtual conjugate particles or from a reconstituted sample of lyophilized virtual conjugate particles.
  • the pharmaceutical composition dose can be calculated based on delivery of a specified mass of virtual conjugate particles, for example 100 pg. in that case, a specific mass of each monovalent virtual conjugate particle composition is measured by weight or volume. In other instances, the dose is based on delivering a specified mass of antigen. For example, 2,2 pg polysaccharide or 10 pg protein, in this case the loading of the antigen on the virtual conjugate particle must be known.
  • PnPs 6A Polysaccharide
  • PnPs 19A Polysaccharide
  • HA hemagglutinin
  • RSV-F respiratory syncytial virus
  • ⁇ PnPs are from Ihe Serum Institute of India (SH) and are native unless otherwise noted.
  • Particles were manufactured utilizing PRINT ⁇ Technology and poly(iaeiide-co-glycolide) polymer. These methods have previously been described in, for example U.S. Pat. os. 8,518,316; 8,444,907: 8,420, 124; 8,268,446; 8,263,129; 8,158,728; 8,128,393; 7,976,759; U.S. Pat. Application Publications Nos.
  • Example 1 was repeated using a FLUOROCUR ' "' 1 Moid with 200 nrn by 200 nm by 200 nm cubical cavities.
  • Example 1 was repeated using poly(lactide-co-glycolide) polymer blended with 10% by weight DC-cholesterol.
  • Example 1 was repeated using poly(lactide-co-glycolide) polymer blended with 3% by weight dimethyiaminoethyi methacrylate (DMAEMA).
  • DMAEMA dimethyiaminoethyi methacrylate
  • a mass of particles fabricated by any of Example 1 , Example 2 or Example 4 were aliquoied into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL
  • a 5% ⁇ 5 mg/mL in water) protamine sulfate solution was added and the suspension was rocked at room temperature overnight.
  • the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded.
  • the pellet was sonicated in 300 to 500 ⁇ water until no particle chunks were present.
  • the volume was increased to 1 ml to continue to wash the particles.
  • the suspension was centrifuged a second time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in water.
  • the suspension was centrifuged a second time at 12,500 X g for 30 minutes.
  • the wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes.
  • the supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in 0.1 % polyvinyl alcohol/5% mannitoi.
  • Protamine and protein were combined in a 0.5 mL tube and allowed to incubate at room temperature for one hour.
  • the protamine/protein mixture was added to an aliquot of the particles of any of Example 1 or Example 2 and water was added to achieve a targeted concentration, for example 2 mg/mL.
  • the suspension was rocked in the tube at room temperature for one hour.
  • To wash away unbound protamine and/or protein the suspension was centrifuged at 12.500 X g for 30 minutes and the supernatant was discarded.
  • the pellet was sonicated in 300 to 500 ⁇ water until no particle chunks were present.
  • the volume was increased to 1 mL to continue to wash the particles.
  • the suspension was centrifuged a second lime at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re- suspended to a targeted concentration, for example 2 mg/mL, in water.
  • the wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in 0.1 % polyvinyl alcohol/5% mannitol.
  • the suspension was centrifuged a second time at 12,500 X g for 30 minutes.
  • the wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes.
  • the supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 rog/mL, in 0.1 % polyvinyl alcohol/5% mannitoi.
  • the suspension was centrifuged a second time at 12,500 X g for 30 minutes.
  • the wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes.
  • the supernatant was discarded and the pellet was re-suspended a targeted concentration, for example 2 mg/rnL, in 0.1 % polyvinyl alcohol/5% mannitoi.
  • Examples 5 through Example 8 The various additions were calculated as described below.
  • the core particle is a suspension while the protamine protein, polysaccharide, protein, and cross-linker are solutions in fabrication. Consequently, in fabrication, unbound protamine protein, polysaccharide, protein, and cross-linker are washed away. Those percentages or masses are noted as fabrication percentages or fabrication masses respectively. The total fabrication mass is noted as such as the value is comprised of the masses of the protamine protein, polysaccharide, protein, and cross-linker during fabrication.
  • the total fabrication mass of the virtual conjugate particles was equal to the sum of the core particle mass plus the protamine protein fabrication mass plus the polysaccharide fabrication mass plus the protein fabrication mass.
  • the total fabrication mass of the virtual conjugate particles was also calculated based on the targeted component percentages for the virtual conjugate particle. Using this method of calculation, the total fabrication mass of the virtual conjugate particles was equal to the core particle mass divided by the core particle mass percent, where the core particle mass percent was equal lo one minus Ihe prolamine protein target fabrication percenl minus the polysaccharide target fab ication percent minus the protein target fabrication percent. Expressed mathematically:
  • Protamine target fabrication mass (mg) (fabrication % protamine) * (total fabrication mass of virtual conjugate particles (mg))
  • Polysaccharide target fabrication mass (mg) (fabrication % polysaccharide) * (total fabrication mass of virtual conjugate particles (mg))
  • Protein target fabrication mass (mg) (fabrication % protein) * (total fabrication mass of virtual conjugate particles (mg))
  • Protamine target volume (ml.) Protamine target fabrication mass (mg) / (concentration protamine stock solution (mg/mL)), to convert to ⁇ _, the result was multiplied by 1000.
  • Polysaccharide target volume (ml.) Polysaccharide target fabrication mass (mg) /
  • Protein target volume (mL) Protein target fabrication mass (mg) / (concentration protein stock solution (mg/mL)), to convert to pL, the result was multiplied by 1000.
  • the mass of the core particles was divided by the core particle mass percent, wherein the core particle mass percent was equal to one minus the protamine target fabrication percent minus the polysaccharide target fabrication percent minus the protein target fabrication percent.
  • the target amount of EDC added was relative to the target fabrication mass of the carrier protein added.
  • the target amounl of EDC added was ten times the target fabrication mass of carrier protein added.
  • the target amount of BMH added was relative to the target fabrication mass of the carrier protein being added.
  • the target amount of BMH added was two times the target fabrication mass of the carrier protein added.
  • Example 3 Particles of Example 3 were washed three times in nuclease-free water. The particles were re-suspended in water to a concentration of 8.3 mg/mL 602 pi particles were combined with 800.2 ⁇ water, and 97.8 ⁇ _ of a 1.1 mg/mL solution of HA-RNA. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 12,000 X g for twenty minutes at 4°C. The resulting pellet was re-suspended in 2 mL of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 12,000 X g for twenty minuies at 4°C.
  • the resulting pellet was re-suspended in 365.5 pL water and 134.4 pi of a 2% prolamine sulfate solution were added. The suspension was rocked for one hour at room temperature. The particles were washed again as described above. The final sample was diluted to 5.37 mg/mL using an aqueous (wt%) 0.2% polyvinyl alcohol/10% mannito! solution.
  • Example 2 125 pL particles of Example 2 were combined with 1 1 15 pL 0.1 % polyvinyl alcohol and 67,2 pi of a 2% protamine sulfate solution. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 10,000 X g for twenty minutes at 4"C and the supernatant was decanted off. The resulting pellet was re- suspended in 2 ml of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 10,000 X g for twenty minutes at 4°C and the supernatant was decanted off.
  • the pellet was re- suspended in 201 pL water and 97.8 pL of a 1.1 mg/mL solution of HA-RNA was added. The suspension was rocked at room temperature for one hour. The sample was diluted to 1 mL using a 0.2% polyvinyl alcohol/10% mannito! solution to a final concentration of 5.48mg/mL
  • Example 2 125 pL particles of Example 2 were combined with 1 1 15 pL 0.1 % polyvinyl alcohol and 87.2 pL of a 2% protamine sulfate solution. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 10,000 X g for twenty minutes at 4°C. The resulting pellet was re-suspended in 2 mL of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 10,000 X g for twenty minutes at 4°C. The pellet was re-suspended in 225 pL water and 51 pL of a 2.1 mg/mL solution of RSV-F-RNA was added. The suspension was rocked at room temperature for one hour. The sample was diluted to 1 mL using 0.2% polyvinyl alcohol/10% mannito! to a final concentration of 5.48 mg/mL.
  • Example 12 Example 12
  • PREVNAR 13 ⁇ is a commercially available pneumococcal conjugate vaccine manufactured by Wyeth Pharmaceuticals, inc. and marketed by Pfizer, inc.
  • the vaccine contains purified capsular polysaccharide from thirteen of the Streptococcus pneumoniae serotypes; PnPsl , PnPs3, PnPs4, PnPsS, PnPs6A, PnPs6B, PnPs7F, PnPs9V, PnPs14, PnPs18C, PnPs19A, PnPs19F, and PnPs23F.
  • each 0.5 mL dose contains approximately 2.2 pg of twelve serotypes and approximately 4.4 pg of serotype PnPs68. in addition, each dose contains approximately 32 pg CRM 97> 125 pg aluminum phosphate (adjuvant), 0.295 mg (buffer) and 0.02% polysorbate 80 (stabilizer).
  • PREVNAR 13 ⁇ is indicated for the active immunization for the prevention of invasive disease caused by the thirteen serotypes in children 6 weeks through 17 years of age (prior to the 18 Th birthday) and in adults 50 years of age and older.
  • PREVNAR 13 ⁇ is also indicated for the aciive immunization for the prevention of otitis media caused by S. pneumonia serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.
  • ⁇ VI ⁇ Typhoid Vi Polysaccharide Vaccine ⁇ Sanofi Pasteur SA is a commercially available vaccine.
  • the vaccine contains the cell surface Vi polysaccharide extracted from Salmonella enterica serovar Typhi, S. typhi Ty2 strain.
  • Each 0.5 mL dose is formulated to contain 25 pg purified Vi polysaccharide in a colorless isotonic phosphate buffered saline (pH 7 ⁇ 0.3), 4.150 mg sodium chloride, 0.065 mg disodium phosphate, 0.023 mg monosodium phosphate, and 0.5 ml sterile water for injection.
  • TYPHIM Vi® is indicated for the active immunization against typhoid fever for persons two years of age or older.
  • Anti-PnP antibodies were diluted to 0.15pg/mL in 0.05M carbonate/bi-carbonate buffer (pH 9.6) and added at 100pL/weil to Maxisorb 98-vve!l plates, which were then sealed and incubated at 4 C overnight. The next morning, the coating solution was decanted and the plates were washed one time using washing buffer (1x PBS, 0.2% Tween-20). Blocking buffer (1x PBS, 2% BSA, 0.2% Tween-20) was added at 200pL/weli. The plates were sealed and incubated at 37 ° C for greater than or equal to two hours. [0100] Samples and standards were prepared during the blocking step.
  • Particle samples and PnP standard were treated with an equal volume of Proteinase K enzyme (PK, stock 1 mg) for 40 minutes at 65 ° C with intermittent vortexing.
  • PK Proteinase K enzyme
  • a 1 :25 dilution of 0.1 M PefaBloc SC Plus was added to all samples and standards. Samples were treated in the order that they would be added to plates in the next step. After the PK treatments, the clarity of samples prior to diluting (cloudy, hazy, clear) was recorded.
  • the treated standard was diluted in blocking buffer to the target starting concentration of 25 ng/mL then serially diluted 6 x 1/2 in blocking buffer. Treated samples were serially diluted 4 x 1/10 in blocking buffer.
  • PNPP substrate tablets were dissolved in ultrapure water (1 gold and 1 silver tablet per 20 ml_s of water) and passed through a 0.2 ⁇ filter. The plates were washed six times with wash buffer. PNPP substrate solution was added at 100 ⁇ _ ⁇ / ⁇ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the highest concentration of the standard curve to have an OD of approximately 1.0-1.5. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm (with purple plate adapter in place). Custom SoftMax Pro templates were used for data analysis.
  • Anti-Vi antibodies were diluted 1 : 1000 in 0.0 5M carbonate/bi-carbonate buffer (pH 9.6) and added at 100 ⁇ _ ⁇ / ⁇ to Maxisorb 96-well plates, which were then sealed and incubated at 4 ° C overnight. The next morning, the coating solution was decanted and the plates washed one time with washing buffer (1x PBS, 0.2% Tween-20). Blocking buffer (1x PBS, 2% BSA, 0.2% Tween-20) was added at 200 ⁇ _ ⁇ / ⁇ . The plates were sealed and incubated at 37 ° C for greater than or equal to two hours.
  • Samples and standards were prepared during the blocking step. Preparation of two standards is required. First, a Vi-CRM GA crosslinked standard was read against all particle groups crosslinked with GA. Secondly, a Vi-CRM EDC crosslinked standard was read against all particle groups crosslinked with EDC. The standards were diluted in blocking buffer to the desired starting concentration of 1000 ng/mL then serially diluted 7 x 1/2 in blocking buffer. Samples were diluted 1/10 in blocking buffer for the first dilution. Then samples were serially diluted 3 x 1 /4 in blocking buffer.
  • IgG IgG was diluted 1 :1000 in blocking buffer. The plates were washed four times using wash buffer. AP-lgG was added at 100 ⁇ _ ⁇ / ⁇ to the plates with either 40 or 60 seconds between each plate to ensure consistency in development. The plates were sealed and incubated at 37 ° C for one hour.
  • PNPP substrate tablets were dissolved in ultrapure water and passed through a 0.2 ⁇ filter. The plates were washed six times using wash buffer. PNPP substrate solution was added at 100 ⁇ _ ⁇ / ⁇ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the highest concentration of the standard curve to have and OD of approximately 2.0. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm (with purple plate adapter in place). Custom SoftMax Pro templates were used for data analysis.
  • Virtual conjugate particles were analyzed for protein content using the Protein Content Assay Bicinchoninic Acid (BCA) Protocol. Formulated particle suspensions were centrifuged to pellet the particles, with the resulting supernatant discarded. The pellets were reconstituted in phosphate buffered saline (PBS) and diluted to a working concentration. BCA Reagent was then added and the samples incubated at 37°C for a minimum of three hours. The resulting samples were then cooled to room temperature and read on a Spectramax M5 microplate reader at 562 nm. Data was analyzed by the instrument software.
  • BCA Protein Content Assay Bicinchoninic Acid
  • Immunological Testing IgG ELISA Protocol [0109] Virtual conjugate particles were analyzed for immunological activity utilizing an ELISA assay.
  • PnPs was diluted to 2 or 20 ⁇ g/mL or PLD was diluted to 2 ⁇ g/mL in 1X PBS (pH 9.6) and added at 50 ⁇ Uwe ⁇ to Maxisorb 96-well plates, which were then sealed and incubated at 4°C overnight. The next morning, the coating solution was decanted and the plates washed one time with washing buffer (1X PBS, 0.2% Tween-20). Blocking buffer (1X PBS, 2% BSA, 0.2% Tween-20) was added at 200 ⁇ UweW. The plates were sealed and incubated at 37°C for greater than or equal to two hours.
  • PLD Samples Samples and controls were prepared during the blocking step. Serum samples were thawed at 4°C overnight. Absorption buffer (0.05% Tween-20, 1X PBS) was prepared. Serum samples were diluted 1/40 in absorption buffer, and control samples were diluted to a predetermined concentration in absorption buffer. Samples were added to the dilution plate in the order than they would be added to plates in the next step. Samples were then diluted 10X 1/2 in absorption buffer. [01 14] The blocking buffer was decanted and the plates washed three times with wash buffer. Samples and controls were added at 50 ⁇ _ ⁇ / ⁇ to the plates in duplicate. The plates were sealed and incubated at 37°C for one hour.
  • Absorption buffer 0.05% Tween-20, 1X PBS
  • the secondary antibody was diluted in blocking buffer.
  • the secondary antibody alkaline-phosphatase-conjugated goat-anti-RABBIT IgG, subclasses 1 +2a+2b+3, F Cy fragment specific
  • the secondary antibody alkaline-phosphatase-conjugated goat-anti-MOUSE IgG, subclasses 1 +2a+2b+3, F Cy fragment specific
  • the plates were washed three times with wash buffer.
  • Mouse PnPs IgG plates were washed four times.
  • the secondary antibody solution was added at 50 ⁇ _ ⁇ / ⁇ to the plates with either 40 or 60 seconds between each plate to ensure consistency in development. The plates were sealed and incubated at room temperature for one hour.
  • PNPP substrate tablets were dissolved in ultrapure water and passed through at 0.2 ⁇ filter. The plates were washed three times with wash buffer. Mouse PnPs IgG plates were washed four times. PNPP substrate solution was added at 100 ⁇ _ ⁇ / ⁇ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the control samples to reach a predetermined optical density. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm. Custom SoftMax Pro templates were used for data analysis.
  • Typhoid Polysaccharide was derived from Nahm M and Goldblatt D. 26 November 2002 posting date, Training manual for enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (PnPg ELISA) and Wernette CM, et. al. 2003. Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides, Clin. Diagn. Lab. Immunol. 10:514-591.
  • Antibody Buffer 10X Stock (10X PBS/0.2% NaN 3 /0/5% Tween 20): 80 g NaCI, 3.14 g KH 2 P0 4 , 20.61 g Na 2 HP0 4 ⁇ 7 H 2 0, 1.6 g KCI, 2 g NaN 3 were dissolved in 1000 mL dH 2 0. 5 mL Tween 20 was added. After dissolution, the solution was sterile filtered and stored at room temperature.
  • Antibody Buffer (1X PBS/0.02% NaN 3 /0/05% Tween 20): 900 mL dH 2 0 was added to 100 mL Antibody Buffer 10X Stock. The pH was determined using an aliquot from the resulting solution. If the pH was not 7.2 +/- 0.2, the Antibody Buffer 10X stock solution was discarded and a new solution was prepared.
  • Wash Buffer 10X Stock 80 g NaCI, 1.6 g KCI, 0.94 g Trizma Base, 14.56 g Trizma HCI were weighed. dH 2 0 was added to 800 mL. After dissolution, 33 mL Brij-35 (30% wt/v) was added and the solution was mixed thoroughly. The final volume was brought to 1000 mL using dH 2 0. The solution was stored at room temperature for up to 12 months.
  • Wash Buffer One part Wash Buffer 10X Stock was combined with nine parts dH 2 0. The pH was verified to be 7.2 +/- using an aliquot of the resulting solution.
  • Stop Solution 120 g NaOH pellets were slowly added to 800 mL dH 2 0 to control the generation of the resulting exotherm. After the dissolution was complete and the solution cooled to room temperature, the total volume was brought to 1000 ml. using dH 2 0. the solution was stored at room temperature for up to 12 months.
  • Vi Tephoid Polysaccharide was diluted to 1 .25 ⁇ g/mL in Coating Buffer and added at 100 iUweW to medium binding, 96-well flat bottom, polystyrene plates (for example Greiner 655001 , or equivalent), which were then covered and incubated at 37 ° C for 5 hours. Plates were then cooled and stored at 4°C overnight until day of the assay (for up to 1 week). On the day of the assay, plates were brought to room temperature until samples were ready to be added.
  • medium binding, 96-well flat bottom, polystyrene plates for example Greiner 655001 , or equivalent
  • Serum samples were thawed at 4°C overnight the night before the assay.
  • Fresh Antibody Buffer was prepared from Antibody Bufferl Ox Stock. Serum samples were diluted 1/40 in Antibody Buffer, and control samples (positive and negative) were diluted to a predetermined concentration in antibody buffer. Samples were added in the order that they would be added to plates in the next step. Samples were then serial diluted two fold ten times in Antibody Buffer across a row.
  • Coating Buffer was then decanted and plates were washed with Wash Buffer. The first wash incubated for 1 minute, then plates were washed four more times for a total of five washes. Plates were then blotted dry on paper towel. Samples and controls were added at 50iUweW to the plates in duplicate. Plates were sealed and incubated at room temperature for approximately two hours.
  • the secondary antibody (alkaline-phosphatase-conjugated goat-anti-mouse IgG, ⁇ -chain specific; for example Sigma-Aldrich No. A3438, or equivalent) was diluted 1 :8000 in Antibody Buffer. Plates were washed as described above for a total of five washes using Wash Buffer. The secondary antibody solution was added at 100 ⁇ _ ⁇ / ⁇ to the plates with two minutes between each plate to ensure consistency in development. The plates were covered and incubated at room temperature for two hours. [0130] Approximately 30 minutes prior to development, PNPP substrate tablets were dissolved in ultrapure water and passed through a 0.2 ⁇ filter.
  • PNPP substrate solution was added at 100 ⁇ _ ⁇ / ⁇ with two minutes between each plate to ensure consistency in development. Plates were incubated at room temperature for two hours.
  • 96 well microtiter plates were coated with a protein solution containing either RSV F or HA protein at a concentration of approximately 2 ⁇ g/mL in carbonate-bicarbonate buffer. Plates were covered and incubated at 4°C overnight. The following morning the coating solution was removed from the plates and the plates were washed in 1X PBS using a plate washer. 200 ⁇ _ Blocking Buffer (30 g BSA + 1 L 1X PBS) was added to all wells and the plates were covered and incubated for approximately 1-2 hours at 37°C.
  • Serum samples and control were prepared during the blocking incubation period. Serum samples were diluted in Reagent Diluent (10 g BSA + 500 ⁇ _ Tween 20 + 1 L 1X PBS) to the desired starting concentration in a minimum total volume of 300 ⁇ _. Samples were serially diluted 40 fold from column 1 to column 2, then serially diluted 2 fold from column 2 through column 1 1. A positive control was prepared by diluting anti-RSVF (monoclonal Ab, stock at approximately 1 mg/mL) 1 : 1000 by adding 10 ⁇ Ab into 10 mL of Reagent Diluent (final concentration approximately 31.3 ng/mL).
  • Reagent Diluent 10 g BSA + 500 ⁇ _ Tween 20 + 1 L 1X PBS
  • Blocking Buffer was removed and the plates were washed three times with 1X PBS using a plate washer. 50 [iL of diluted serum sample per well was transferred to the ELISA plate. Samples were run in duplicate. The plate was covered and incubated at 37°C for approximately one hour.
  • Secondary antibody was prepared at a dilution of 1 :1000 by adding 200 ⁇ Ab into 200 mL of Assay Diluent (AP-conjugated Goat Anti-mouse IgG (subclasses 1 + 2a + 2b + 3), Fc- ⁇ fragment specific (Jackson I mmu no Research catalog 115-055-164)). Plates were washed three times with 1X PBS using a plate washer. The plates were blotted dry. 50 ⁇ Uwe ⁇ of secondary antibody was added to each plate at 40 second intervals and the plates were incubated at room temperature for approximately one hour.
  • Assay Diluent AP-conjugated Goat Anti-mouse IgG (subclasses 1 + 2a + 2b + 3), Fc- ⁇ fragment specific (Jackson I mmu no Research catalog 115-055-164). Plates were washed three times with 1X PBS using a plate washer. The plates were blotted dry. 50 ⁇ Uwe ⁇ of secondary antibody was added to
  • Red Blood Cell Lysis Solution by dissolving approximately 8.29 g ammonium chloride, 1.0 g potassium bicarbonate, and 0.0372 disodium EDTA dehydrate in 900 mL deionized water. Adjust the pH to 7.2 to 7.4 using sodium hydroxide. Add deionized water to a final volume of 1 L and sterile filter. Store at room temperature.
  • 70% ethanol was prepared from 100% ethanol using sterile water. Coating solutions were prepared by diluting mouse IL-2 (interleukin-2) 1 :200 with sterile 1X PBS (0.275 mL into 54.725 mL) and by also diluting mouse IFN-gamma (interferon-gamma) 1 :200 with sterile 1X PBS (0.275 mL into 54.725 mL).
  • Membranes of the MultiScreen-IP Hydrophobic PVDF 96 well plates were pre-wetted by adding 15 ⁇ Uwe ⁇ of 70% ethanol for 50-60 seconds. The ethanol solution was decanted and the plates were washed three times with 1X PBS using 150 ⁇ UweW. Next the plates were coated by adding 100 ⁇ of the appropriate coating solution and incubate overnight at 4°C.
  • the coating solution was decanted and the plates were washed three times with cRPMI medium (10% fetal bovine serum/1 % Pen/strep/1 % nonessential amino acids/1 % sodium pyruvate/25 mM Hepes/0.1 % BME) leaving the last wash as a blocking medium.
  • the plates were then incubated at room temperature for at least two hours.
  • the splenocytes were prepared. Spleens were mashed gently and spun at 450 X g at room temperature for approximately 5 minutes. The medium was removed and the tube scraped to loosen the cells. Next, 10 mL of RBC lysis solution was added to the tube and the tube was inverted several times. The tube was spun again at 450 x g at room temperature for approximately 5 minutes. The supernatant was removed and the tube scraped to loosen the cells. Next, 15 mL of cRPMI medium was added. The suspension was poured through a 40 ⁇ cell strainer into a sterile 50 mL conical tube. The filtered suspension was transferred into a fresh 15 mL conical tube and centrifuged a final time at 450 x g at room temperature for approximately 5 minutes. The medium was decanted leaving approximately 0.5 to 1.0 mL medium with the cells.
  • the trypan blue cell stain was prepared by adding 2 ⁇ of the splenocytes to 498 ⁇ of 0.1 % trypan blue. The cells were counted in each of the four corner quadrants of the hemacytometer. Also, based on cell count 3e6 cells/mL to a total volume of 3.5 mL for each sample were prepared.
  • Various stimulation media were prepared.
  • Working solutions of PMA, ionomycin, and nucleic acid peptide were prepared.
  • PMA a stock solution of approximately 2 mg/mL was diluted 1 :40 in cRPMI medium by adding 10 ⁇ of stock PMA into 390 ⁇ of cRPMI.
  • ionomycin a stock solution of approximately 10 mM and 7.14 mg/mL was diluted 1 :10 in cRPMI medium by adding 15 ⁇ of stock into 135 ⁇ of cRPMI.
  • For HA-RNA peptides vials were prepared at approximately 100 ⁇ g/mL.
  • RSV-F RNA peptide vials were prepared at approximately 100 ⁇ g/mL.
  • Positive stimulation medium was prepared by combining approximately 30 ⁇ working solution of PMA, 30 ⁇ working solution of ionomycin, 30 ⁇ of alpha CD28, and 2.1 mL of cRPMI medium.
  • Negative stimulation medium was prepared by combining approximately 50 ⁇ of alpha CD28 and 4.95 mL of cRPMI medium.
  • HA RNA peptide stimulation medium was prepared by combining approximately 0.6 mL of working HA-RNA peptide solution, 30 ⁇ _ alpha CD28, and 2.37 mL cRPMI medium.
  • RSV-F RNA peptide stimulation medium was prepared by combining approximately 0.2 mL of working RSV-F RNA peptide solution, 10 ⁇ alpha CD28, and 0.79 mL cRPMI medium.
  • Buffers were prepared. Wash buffer I was prepared by adding 0.05% Tween 20 to 1X PBS. Wash buffer II was 1X PBS. The assay diluent was 1X PBS with 10% FBS.
  • the cell suspension was decanted from the plates. The plates were washed two times using deionized water with a 3-5 minute soak for each water wash. Next, the plates were washed three times with wash buffer I.
  • the detection antibody (Anti IL-2 and Anti IFN gamma) was diluted 1 :250 in assay diluent (10% fetal bovine serum in 1X PBS)by adding 0.22 mL of antibody into 54.88 mL of buffer for each cytokine. The detection antibody was added at 100 ⁇ Uwe ⁇ and the plates were covered and allowed to incubate at room temperature in the dark for approximately two hours.
  • substrate solution was prepared by adding 1 drop (approximately 30 ⁇ ) of AEC Chromogen per 1 mL of AEC substrate. The substrate solution was vortexed thoroughly and stored in the dark at room temperature until use.
  • Plates were removed from the dark and the HRP solution discarded. The plates were washed four times with wash buffer 1 and two times with wash buffer II. To develop plates, 100 iUweW of substrate solution was added. The plates were allowed to develop until the desired color was achieved, generally 30-60 minutes. Once the desired color was achieved the plates were submerged in deionized water. The plastic backing was then removed and the plates were re-submerged in fresh deionized water. Excess water was removed and the plates were dried overnight in the dark.
  • a series of virtual conjugate particles were produced substantially according to Example 5 for injection into mice. For this study, approximately 100 ⁇ g of the pharmaceutal composition in approximately 500 ⁇ _ vehicle was injected. Injections were placed in the dorsal thoracic region using a 21 gauge needle. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
  • Samples were produced using the core particle of Example 1 (PLGA, 80 nm X 320 nm). All samples for this study were cross-linked utilizing glutaraldehyde.
  • a mass of particles fabricated by any of Example 1 were aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL
  • a 5% (5 mg/mL in water) protamine sulfate solution was added and the suspension was rocked at room temperature overnight.
  • the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded.
  • the pellet was sonicated in 300 to 500 ⁇ _ water until no particle chunks were present.
  • the volume was increased to 1 ml. to continue to wash the particles.
  • the suspension was centrifuged a second time at 12,500 X g for 30 minutes.
  • the supernatant was discarded and the pellet was re- suspended to a targeted concentration, for example 2 mg/mL, in water.
  • the polysaccharide content was determined according to Example 13.
  • the protein content was determined according to Example 14.
  • the table below contains the test results.
  • FIG. 8 shows the Anti-PnPs14 IgG Reciprocal Titer for each sample at four points in time: pre-bleed, post-prime, post-boost 1 , and post-boost 2.
  • Sample 51-15 was primed one week after the other two groups, so post-prime data was collected at three weeks instead of four weeks like the other samples.
  • each pharmaceutical composition produced had immunological activity greater than or equal to that of PREVNAR 13®.
  • the pharmaceutical composition showed immunological activity approximately equivalent to PREVNAR 13® when PnPs14 was combined with OVA, CRM 197 , or PLD when using glutaraldehyde as the cross-linker.
  • a series of virtual conjugate particles were produced for implantation into mice. For this study, approximately 100 ⁇ g of the pharmaceutal composition in approximately 500 ⁇ _ vehicle was injected. Injections were placed in the dorsal thoracic region using a 21 gauge needle. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
  • Samples 55-6 through 55-1 1 , 55-13, and 55-14 utilized the core particle of Example 3 (PLGA/DC-Chol).
  • Samples 55-15 through 55-26 utilized the core particle of Example 1 (PLGA, 80 nm X 320 nm). This animal study examined various polysaccharides, cross-linkers, and processing methods.
  • Samples 55-7 through 55-1 1 were produced according to Example 7.
  • Samples 55-18 through 55-23 were produced according to Example 5.
  • Sample 55-13 and 55-24 were produced substantially according to Example 8 and Example 6 respectively. When removing the unbound protamine and protein, the particles were washed once instead of twice.
  • Samples 55-14 and 55-25 were produced substantially according to Example 7 and Example 5 respectively. Instead of adding glutaraldehyde as described in Examples 7 and 5, bismaleimidohexane (in DMSO) was added to the suspension. After agitation for one hour, the suspension was filtered with a 1 1 ⁇ filter to remove any crystalized BMH. The virtual conjugate particles were then washed twice as described.
  • Samples 55-6 and 55-15 were produced as follows. A mass of particles fabricated by Example 1 (55-15) or Example 3 (55-6) was aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL Protamine was added to 55-15. Polysaccharide and protein were added to the particle suspension using stock solutions. The suspension was agitated for one hour at room temperature. No cross- linker was used. The resulting virtual conjugate particles were washed twice as described above.
  • Samples 55-16 and 55-17 were produced substantially according to Example 5. However, prior to washing the particles after the polysaccharide and protein additions, additional protamine was added to the suspension. The suspension was agitated for one hour at room temperature and the resulting virtual conjugate particles washed twice as described above. Sample 55-17 was then cross-linked using glutaraldehyde as follows. Glutaraldehyde solution was added to the re-suspended particles and agitated for one hour at room temperature. Sodium cyanobrohydride was added to quench the reaction and the virtual conjugate particles were washed twice as described above. [0168] Sample 55-26 was produced as follows.
  • a mass of particles fabricated by Example 1 were aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL Protamine solution was added and the suspension was incubated at room temperature for ten minutes. Polysaccharide was added and the suspension was incubated at room temperature for ten minutes. Protein was added and the suspension was incubated at room temperature for ten minutes. The remaining protamine was added and followed immediately with the addition of glutaraldehyde. After agitation for one hour at room temperature, the glutaraldehyde was neutralized with sodium borohydride and the resulting virtual conjugate particles washed twice as described above.
  • Protamine solution was added and the suspension was incubated at room temperature for ten minutes. Polysaccharide was added and the suspension was incubated at room temperature for ten minutes. Protein was added and the suspension was incubated at room temperature for ten minutes. The remaining protamine was added and followed immediately with the addition of glutaraldehyde.
  • the polysaccharide content was determined according to Example 13.
  • the protein content was determined according to Example 14.
  • the table below contains the test results.
  • Figure 9 shows Anti-PnPs1 IgG Reciprocal Titer for samples utilizing the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm).
  • glutaraldehyde (55-7) produced a higher titer than EDC (55-13) which produced a higher titer than BMH (55-14).
  • PLD 55-7
  • CRM 197 55-9
  • sized polysaccharide When comparing sized polysaccharide to native polysaccharide, sized polysaccharide (55-8) provided a higher titer than native polysaccharide (55-7) for PnPsl , PLD carrier protein, and glutaraldehyde cross-linker. Also of note, the non-cross-linked sample (55-6) produced a lower titer than the control.
  • glutaraldehyde was an acceptable cross-linker with either PLD or CRM 197 as a carrier protein. Using sized polysaccharide further boosted the titer response.
  • Figure 10 shows Anti-PnPs1 IgG Reciprocal Titer for samples utilizing the particle of
  • Example 1 (PLGA, 80 nm X 320 nm).
  • EDC 55-24
  • glutaraldehyde 55-
  • Figure 9 and Figure 10 establish that while the various combinations disclosed herein are effective, certain combinations of carrier proteins and cross-linkers provide particles that induce a greater titer. For example, for PnPsl , the PLGA protamine particle provided a greater titer response than the PLGA DC-Choi particle.
  • Figure 1 1 shows Anti-PnPs5 IgG Reciprocal Titer for samples 55-10, 55-1 1 , 55-22, and 55-23.
  • the use of native and sized PnPs5 polysaccharide was examined.
  • the carrier protein was PLD and the cross-linker was glutaraldehyde.
  • both native (55-10) and sized (55-11 ) PnPs5 performed approximately equal to PREVNAR 13® control.
  • both native (55-22) and sized (55-23) performed approximately equal to PREVNAR 13® control.
  • the purpose of this study was to evaluate the efficacy of a trivalent composition.
  • three separate groups of particles each containing a different serotype were manufactured and then combined to produce a trivalent composition.
  • To produce the trivalent composition a volume containing approximately 100 ⁇ g of virtual conjugate particles from each of the various serotype compositions are combined. These samples were injected subcutaneously in the dosal thoracic region.
  • Sample 56-10 utilized the particle of Example 3 (PLGA DC-Chol, 80 nm X 320 nm) and followed Example 7.
  • Sample 56-18 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5.
  • Sample 56-25 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5 for PnPs5 (sized) and PnPs14 (native) and followed Example 6 for PnPsl (sized).
  • the various serotypes were formulated into a trivalent composition by combining the various serotypes. A volume containing approximately 100 ⁇ g of virtual conjugate particles from each of the various serotype compositions were combined.
  • a control sample (56-03) using protein only was prepared by dispersing PLD in 0.1 % polyvinyl alcohol/5% mannitol at a concentration of 0.012 mg/0.50 ml_.
  • Immunological activity was evaluated according to Example 15. The immunological activity was evaluated for each serotype independently (PnPsl , PnPs5, and PnPs14) as well as the immune response against PLD. All samples utilized PLD as the carrier protein, sized PnPsl polysaccharide, sized PnPs5 polysaccharide, and native PnPsl 4 polysaccharide.
  • Figure 12 shows Anti-PnPs1 IgG Reciprocal Titer for the three samples.
  • PnPsl the trivalent composition using the EDC cross-linker for PnPsl (56-25) produced the highest titer level.
  • the trivalent composition using glutaraldehyde and the PLGA protamine particle (56-18) produced titer equivalent to that of the PREVNAR 13® control.
  • the trivalent composition using glutaraldehyde and the PLGA DC-Choi particle (56-10) produced an immunological response that was below that of the control.
  • Figure 13 shows Anti-PnPs5 IgG Reciprocal Titer for the three samples.
  • the trivalent composition using the EDC cross-linker for PnPsl again produced the highest titer level, just slightly above that of the PREVNAR 13® control.
  • the trivalent composition using glutaraldehyde and the PLGA protamine particle (56-18) produced titer slightly below than that of the control.
  • the trivalent composition using glutaraldehyde and the PLGA DC-Chol particle (56-10) produced an immunological response that was below that of the control.
  • Figure 15 shows Anti-PLD IgG Reciprocal Titer for the three samples.
  • the soluble PLD control produced the highest titer level.
  • the trivalent composition using the EDC cross-linker for PnPsl (56-25) again produced the highest titer level, although below that of the soluble control.
  • the two glutaraldehyde particles showed approximately equivalent immune response.
  • a series of virtual conjugate particles were produced for implantation into New Zealand White rabbits. Samples were injected via two routes: subcutaneous (SC) and intramuscular (IM). The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
  • Samples 57-06 and 57-07 utilized the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm) and followed Example 7.
  • Samples 57-08 and 57-09 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5.
  • Sample 57-05 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5 for both PnPs5 (sized) and PnPsl 4 (native) and followed Example 6 for PnPsl (sized).
  • the various serotypes were formulated into a trivalent composition by combining the various serotypes based on delivering a targeted dose of antigen.
  • the polysaccharide content for the virtual conjugate particle samples were analyzed according to Example 13. Once the polysaccharide content was known, the composition was prepared to deliver a target dose of 2.2 ⁇ g per serotype.
  • Samples administered via subcutaneous injection included 57-01 , 57-05, 57-06, and 57- 08. Subcutaneous injections were delivered to the dorsal thoracic region. A total of 1.0 mL was delivered using a 21 gauge needle. Samples administered via intramuscular injection included 57-02, 57-07, and 57-09. Intramuscular injections were delivered in the quadriceps muscles located on the cranial aspect of the femur or the hamstrings on the caudal aspect of the femur using a 23 gauge needle. A total of 1.0 ml. was delivered.
  • Figure 16A shows Anti-PnPs1 OPK Reciprocal Titer and Figure 16B shows Anti-PnPs1 IgG Reciprocal Titer
  • Figure 17A shows Anti-PnPs5 OPK Reciprocal Titer
  • Figure 17B shows Anti-PnPs5 IgG Reciprocal titer
  • Figure 18A shows Anti-PnPs14 OPK Reciprocal Titer
  • Figure 18B shows Anti-PnPs14 IgG Reciprocal Titer.
  • SC (57-06) produced an immune response greater than that of IM (57-07) for PLGA DC-Choi based particles while SC (57-08) produced an immune response greater than or equal to that of IM (57-09) for PLGA/protamine based particles.
  • Samples 59-04 and 59-05 utilized the core particle of Example 3 (PLGA/DC-Chol) and were fabricated according to Example 7.
  • Samples 59-14, 59-15, 59-16, 59-21 , and 59-22 utilized the core particle of Example 1 (PLGA, 80 nm X 320 nm).
  • Sample 59-14 was fabricated substantially according to Example 5 with the omission of glutaraldehyde.
  • Samples 59-15 and 59-16 were fabricated according to Example 5.
  • Samples 59-21 and 59-22 were fabricated according to Example 6.
  • Sample 59-13 utilized the particle of Example 4 (PLGA DMAEMA). All samples were lyophilized with the exception of 59-04 and 59-15. Lyophilized samples were reconstituted in 0.1 % polyvinyl alcohol/5% mannitol prior to inoculation at a target concentration of 0.3 mg/0.5 ml_.
  • the polysaccharide content was determined according to Example 13.
  • the protein content was determined according to Example 14.
  • the table below contains the test results.
  • Figure 19 shows Anti-Vi IgG Reciprocal Titer for the tested samples that included protamine.
  • Figure 20 shows Anti-Vi IgG Reciprocal Titer for the tested samples that included DC-Choi or DMAEMA.
  • the virtual conjugate particles proved to be a viable composition to deliver typhoid polysaccharide.
  • a series of virtual conjugate particles were produced for implantation into mice.
  • the table below lists the samples IDs and the source of the samples. Five animals were provided for each sample.
  • Figure 21A-C shows the results of the immunological testing for the HA-RNA containing samples.
  • Figure 21 A shows the T cell responses (IL2) in spots per million cells.
  • Figure 21 B shows the T cell responses (IFNg) in spots per million cells.
  • Figure 21 C shows the Anti-Flu HA IgG ELISA Titer for the tested samples.
  • Figure 22A-C shows the results of the immunological testing for the RSV-F RNA containing samples.
  • Figure 22A shows the T cell responses (IL2) in spots per million cells.
  • Figure 22B shows the T cell responses (IFNg) in spots per million cells.
  • Figure 22C shows the Anti-RSV-F IgG ELISA Titer for both the prime and boost samples.
  • the data showed robust T cell responses (IFNg and IL2) for all samples tested.
  • the data also showed humoral responses for all samples tested.
  • the virtual conjugate particles proved to be a viable composition to deliver nucleic acids such as RNA.
  • a series of virtual conjugate particles were produced for implantation into mice.
  • the table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
  • Samples 01 , 02, and 03 provided controls for the study: 01 : PREVNAR®, 02: soluble PLD/PnPs 6A/PnPs 14A, and 03: soluble PLD/PnPs6A/PnPs 14A and Alydrogel.
  • Sample 33 utilized the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm). Particles were produced using either of following procedures.
  • Protamine and PLD were admixed and incubated at room temperature for 1 hour. The admixture was combined with the particles of Example 3 and rocked at room temperature for 1 hour. While the particles were rocking, PnPs 6A or PnPs 19A was combined with ADH and half the volume of EDC for 20 minutes. After 1 hour of rocking, the particles/protamine/PLD was pelleted to recover the protamine/PLD particles. The PnPs/ADH/EDC mixture was added to the pelleted protamine/PLD particles and the remaining volume of EDC was added. The mixture was combined using a pipet for 2 minutes. The mixture was brought to volume using buffer and the reaction was allowed to proceed for 2 hours at room temperature. After 2 hours, the resulting particles were pelleted and washed a total of three times using water (4 total spins).
  • Adipic dihydrazide (ADH) and sodium cyanoborohydride (NaCNBH 3 ) were added to the polysaccharide and the mixture was incubated at room temperature for 1 hour. PLD and the
  • PnPs mixture were added to pelleted protamine particles. EDC was added and the mixture was brought to volume with buffer. The reaction proceeded overnight at room temperature. The resulting particles were pelleted and washed a total of three times with water (4 spins).
  • FIG. 23 shows the response at four points in time: pre-bleed, post-prime, post- boost 1 , and post-boost 2.
  • Anti-PnPs 6A responses are showin in Figure 23A, and anti-PnPs 19A responses are shown in Figure 23B.

Abstract

Virtual conjugate particles containing an active agent such as an antigenic polysaccharide or a nucleic acid with a charged biomolecule such as a protamine protein are described. The virtual conjugate particles typically contain a polymeric core particle that provides a surface for the association of the agent with a charged biomolecule. A cross-linker may be used to associate the agents with the core particle.

Description

VIRTUAL CONJUGATE PARTICLES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001 ] This application claims priority to U.S. Provisional Application No. 61/904,703, filed November 15, 2013, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure is generally directed to pharmaceutical compositions that may be used in a vaccine. Methods for making the pharmaceutical compositions are also described.
BACKGROUND
[0003] Conjugate vaccines are typically created by covalently attaching an antigen to a carrier protein. Conjugation is often applied to bacterial polysaccharides for the prevention of bacterial diseases. Conjugation relies on specific chemistries to link the bacterial polysaccharide to the carrier protein. The conjugation process is complex and involves multiple steps. Both the protein and polysaccharide have to be manufactured, most likely including isolation and purification steps. In some cases, the polysaccharide needs to be sized. For an effective reaction, cierivatization may be required prior to chemical conjugation. After conjugation the composition must be purified to remove unreacfed materials. Some pathogens have multiple serotypes, for example pneumonia. A conjugation procedure that may be optimized with one serotype, may not work with another serotype of the same pathogen. And in some cases, the antigen is not amenable to the conjugation process at all.
[0004] As the conjugation process is a chemical reaction, the relationship between antigen and carrier protein is a stoichiometric one. For example, in U.S. Patent Application Publication 2010/0316666 to Hausdorfi et. ai. the conjugation of serotypes of Streptococcus pneumonia with CRM is disclosed. The table below details the conjugation ratios for the various serotypes.
Figure imgf000003_0001
[0005] Therefore, a pharmaceutical composition where an antigen and a protein are present in appropnale spatial associalion which does not require the direct and difficult conjugation of protein to antigen would be highly desirable. Additionally, a pharmaceutical composition where antigen and protein can be combined in non-sioichiometric ratios would also be highly desirable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] Reference is made to the accompanying drawings which show illustrative embodiments of the presenl irfverftiorf and which should be read in connection with the description of the invention.
FIG. 1 depicts a virtual conjugate particle of the invention.
FIG. 2 depicts a second embodiment of a virtual conjugate particle of the invention.
FIG. 3 depicts a third embodiment of a virtual conjugate particle of the invention. FIG. 4 depicts a fourth embodiment of a virtual conjugate particle of the invention.
FIG. 5 depicts a one pot method of making a virtual conjugate particle of the invention.
FIG. 6 depicts a sequential method of making a virtual conjugate particle of the invention.
FIG. 7 depicts an admixture method of making a virtual conjugate particle of the invention.
FIG. 8 depicts the Anti-PnPs14 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #1 ).
FIG. 9 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
FIG. 10 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
FIG. 1 1 depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #2).
FIG. 12 depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
FIG. 13 depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3). FIG. 14 depicts the Anti-PnPs14 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
FIG. 15 depicts the Anti-PLD IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #3).
FIG. 16A depicts the Anti-PnPs1 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
FIG. 16B depicts the Anti-PnPs1 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study). FIG. 17A depicts the Anti-PnPs5 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
FIG. 17B depicts the Anti-PnPs5 IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
FIG. 18A depicts the Anti-PnPs14 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a rabbit model (Pneumococcal Polysaccharide Rabbit Study).
FIG. 18B depicts the Anti-PnPs14 IgG Reciprocal Titer and the Anti-PnPs14 OPK Reciprocal Titer of virtual conjugate particles produced according to the invention in a rabbit model (Pneumococcal Polysaccharide Rabbit Study). FIG. 19 depicts the Anti-Vi IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Typhoid Mouse Study).
FIG. 20 depicts the Anti-Vi IgG Reciprocal Titer of virtual conjugate particles produced according to the invention tested in a mouse model (Typhoid Mouse Study).
FIG. 21 depicts ELISpot and Anti-Flu HA IgG ELISA Titer for virtual conjugate particles produced according to the invention tested in a mouse model (RNA Mouse Study).
FIG. 22 depicts ELISpot and Anti-RSV-F IgG ELISA Titer for virtual conjugate particles produced according to the invention tested in a mouse model (RNA Mouse Study).
FIG. 23A depicts Anti-PnPs 6A titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #4).
FIG. 23B depicts Anti-PnPs 19A titer of virtual conjugate particles produced according to the invention tested in a mouse model (Pneumococcal Polysaccharide Mouse Study #4).
ABBREVIATIONS
ADH: adipic dihydrazide
BCA: bicinchoninic acid
BMH: bismaleimidohexane
BSA: bovine serum albumin
CRM 197 or CRM197: genetically detoxified mutant of diphtheria toxin cRPMI: complete Roswell Park Memorial Institute medium CWPS/22F: cell wall polysaccharide/22F DC-Chol: cholesteryl 3 -N-(di-methyl-amino-ethyl)-carbamate, DC-cholesterol
DMAEMA: dimethylaminoethyl methacrylate
EDC: 1 -ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme-linked immunosorbert assay
FBS: fetal bovine serum
g: gravity
GA: glutaraldehyde
HA-RNA: self-replicating RNA encoding for HA (hemagglutinin) RNA
HRP: streptavidin-horseradish peroxidase
IgG: immunoglobulin G
ml_: milliliters
OPK: opsonophagocytic killing assay
OVA: ovalbumin
PBS: phosphate buffered saline
PK: proteinase K enzyme
PLD: genetically detoxified mutant of pneumolysin toxin
PLGA: poly(lactide-co-glycolide) polymer PMA: phorbol 12-myristate 13-acetate
PnP or PnPs: purified pneumococcal polysaccharide
PNPP: p-nitrophenyl phosphate
RSV-F RNA: self-replicating RNA encoding for RSV-F (respiratory syncytial virus protein F) RNA
DETAILED DESCRIPTION
[0007] Provided herein are virtual conjugate particles, which contain multiple-layered particles typically comprising a core particle. The virtual conjugate particle approach described herein provides an adaptable p!atform for producing medicines for the treatment and prevention of disease.
[0008] As described herein, multiple pharmaceutical compositions have been fabricated and/or contemplated in the form of particle based and spatial association of active agents, resulting in highly effective pharmaceutically active products, including multiple vaccines and other therapeutic treatments. The pharmaceutical composition generally includes an active agent associated with the particle and a highly charged protein through reacting a cross-linker with the particle, active agent, and charged protein. The pharmaceutical composition may also contain a carrier protein, antigen substance, antigenic protein, adjuvant(s) such as alum (i.e. AlydrogeL Adjuphos), and/or therapeutics.
[0009] In general, the pharmaceutical composition of the present invention comprises: i) a polymeric core particle,
ii) an active agent such as, for example, an antigen, drug, adjuvant, or biologic, and in some embodiments a polysaccharide or a nucleic acid, iii) a charged biomoiecuie such as a protamine protein,
iv) a cross-linker, and v) a carrier protein or an antigenic protein. Virtual Conjugate Particles
[0010] Virtual conjugate particles of the invention are produced by association of an active agent (for example, an antigenic polysaccharide or a nucleic acid) with a charged biomoiecuie (for example, a protamine protein). The virtual conjugate particles typically contain a polymeric core particle that provides a surface for the association of the agent with the charged biomoiecuie.
[0011] In some aspects, a cross-linker may be used to covalently cross-link one or more particle components. For example, the cross-linker may link the charged biomoiecuie (e.g. prolamine) to itself, allowing the particle to physically entangle the agent (e.g., a polysaccharide). Typically, physical entanglement does not include a covaleni interaction. Optionally, physically entanglement may not include an electrostatic interaction.
[0012] In other aspecis, the cross-linker may link the charged biomoiecuie to Ihe agent. In yet other aspects, the cross-linker may covalently link a virtual conjugate particle component to a carrier protein. Cross-linking one or more components to each other may provide for enhanced stability of the virtual conjugate particle.
[0013] In certain aspects, the virtual conjugate particles are prepared so as to prevent cross- linking of certain components. For example, in some aspects, the agent (e.g. polysaccharide or protein antigen) and carrier protein are not linked covalently to each other.
[0014] The virtual conjugate particles are especially useful where the active agent is an antigen.
Virtual conjugation places antigens in appropriate spatial orientation with protein without requiring covaient bonding found in conjugation-based approaches. By avoiding conjugation of individual antigens to individual carriers, the virtual conjugation approach process is not limited to having a required sloichiometry between two molecules. Virtual conjugation therefore provides advantages over old approaches in the art. For example, virtual conjugation approaches disclosed herein are less sensitive to polysaccharide sizing limitations. Conjugation to a carrier can require that an antigen, for example a polysaccharide, is appropriately sized so as to achieve efficient conjugation to a carrier moiety. Thus, in some conjugate vaccines, the number of saccharide repeats in a polysaccharide may not be optimal for inducing an immune response, in contrast, the virtual conjugation approaches herein facilitate enhanced immune responses by providing the option of administering particles containing larger polysaccharides.
[0015] When a polymeric core particle is present, the surface can be used to adsorb the antigen or provide a scaffold. The polymeric core particle surface area is high and can be modulated by the size and/or configuration of the polymeric core particle. Multiple copies of an antigen can be associated with the surface of a single core particle. The number of copies is not bounded by stoicbiometry.
[0016] Use of a polymeric core particle simplifies manufacturing and provides a versatile virtual conjugate particle system. Through the use of stock solutions, the polymeric core particle is surface decorated or surface coated with antigen(s) and/or protein{s) of interest. A cross-linker associates the materials with the surface of the polymeric core particle in a non-stoichiometric ratio. The use of stock solutions allows layering of antigen(s) and/or protein(s) of interest. No modification, such as derealization, is necessary for either the antigen or the protein prior to addition. Selection of an antigen/prolein combination is not dictated by conjugation chemistries. An antigen can be combined with a protein of choice at a ratio of choice. The ratio is not bound by conjugation chemistry or stoichiometric ratios. Lastly, recovery of the virtual conjugate particle is achieved through processes such as centrifugation, washing, or tangential flow filtration. [0017] Microparticles and/or nanoparticies are known in the art. Various types include those produced from microemu!sions including oil in water (Q/W), water in oil (W/O), and water in oil in water (W/O/W) microparticles. Other types of particles include beads/microbeacls/microspheres such as those constructed of various polymers such as polystyrene, functionaiized polystyrene, polymethylmethacrylate, poly( -caproiactone), polylactide, or poiyiiactide-co-glycolide) polymer to name a few.
[0018] Microparticles and/or nanoparticies fabricated through PRINT© Technology (Liquidia Technologies, inc., or svilie, North Carolina) offers microparticles and/or nanoparticies with control over their chemical composition, particle size, particle shape, surface functionality, and other physical and chemical characteristics. Use of this technology in the present invention allows for the co-packaging of antigen(s) and/or carrier proteinics} to stimulate an immune response without the use of conjugation. Particles manufactured utilizing PRINT© Technology have previously been described in, for example, US 8,263, 129: US 8, 128,393; US 7,976,759: WO 2008/1 18861 : WO 2009/1 1 1588: US 2009-0165320: US 2007-0275193; US 2007-0264481 ; WO 2008/127455: US 2008-0181958; WO 2008/106503; and WO 2009/132206; each of which is incorporated herein by reference in its entirety.
[0019] Use of a core particle provides a large surface for the association of the active agent, charged biomoiecule, and carrier protein. The table below details the molecular weights for some exemplary core particles, antigenic proteins, and polysaccharides. The weight of the core particle is several orders of magnitude greater than that of any of the active agents and/or any of the carrier proteins/antigenie proteins. The surface of the very large core particle provides a very large area for association of numerous molecules of active agent, carrier protein, and/or antigenic protein. The association is not bound by a specific stoichiometric value such as that found in the conjugation process. Material Approximate Molecular Weight, kDa
Core Particles
80 X 80 X 320 nm rectangular PLGA Core Particle 1.85 X 10s
1 X 1 X 1 μιη rectangular PLGA Core Particle 1.51 X 109
10 X 10 X 10 μηη rectangular PLGA Core Particle 2.1 1 X 1012
200 X 200 nm (R X H) cylindrical PLGA Core Particle 2.46 X 107
80 X 180 nm (R X H) cylindrical PLGA Core Particle 4.09 X 10s
200 X 600 nm (R X H) cylindrical PLGA Core Particle 9.65 X 107
Carrier Proteins/Antigenic Proteins
CRM197 58.4
PLD 52.8-53
OVA 45
PspA 67-99
TT 150
Active Agents
PnPs 1 (native) 979-2,102
PnPs 1 (sized) 156 PnPs 5
PnPs6A
PnPs 14 (native) 787-1 ,212
PnPs 14 (sized) 138
PnPs 19A
PnPs 23F 5,465
Typhoid 247
RSV-F
Core Particle
[0020] Suitable materials for use in the invention to produce core partic!es comprise a biocompatible polymer. In some embodiments, the polymer is selected from the group consisting of a polyester, a polyanhydride, a polyamide, a phosphorous-based polymer, a poiy(cyanoacrylate), a polyorthoester, a polyurethane, a polyorthoester, a poiyether, a carbohydrate, a polypeptide, a hydroxypropyicelluiose, a poly(ethylene glycol), a wax, a hydrogel, a phosphatidylcholine, a poiydihydropyran, a polyacetal, a biodegradable polymer, and combinations thereof, in some embodiments, the polyester is selected from the group consisting of po!y!acUc acid, po!y!acUde, po!yglyco!ic acid, poly(hydroxybulyrate), polyis- caprolactone), poly^-L-malic acid), polydioxanone, poly{lactide-co-glycolide) polymer, and poiyhydroxyalkanoaie. in some embodiments, the polyanhydride is selected from the group consisting of poiyisebacic acid), poly(adipic acid), and po yiterephtbalic acid), in some embodiments, the po!yamide is selected from the group consisting of po!y(Smino carbonates) and polyaminoacids. In some embodiments, the phosphorous-based poiymer is selected from the group consisting of polyphosphate, a polyphosphonate, and a polyphosphazene.
[0021] Preferably the polymer is po!ylactide, polyglycolic acid, or poly(iactide~co~giycolide) polymer. More preferably the polymer is poiyiactide or poly(iactide-co-glycolide) polymer. Most preferably the polymer is poiyilactide-co-glycolide) polymer.
[0022] The particle may also be comprised of a protein. Such particles and methods to make such particles are described in WO2008/106503 which is incorporated herein by reference in its entirety.
Active Agent
[0023] The virtual conjugate particles of the present invention comprise one or more active agents useful for exerting a biological effect on a subject. Suitable active agents include, for example, drugs, small molecules, proteins, nucleic acids, oligonucleotides, natural oligosaccharides, synthetic oligosaccharides, polysaccharides, adjuvants, antibodies, antibody fragments, and biologies.
[0024] The active agent used in the present invention is preferably a polysaccharide or a nucleic acid. Suitable polysaccharides for use in the invention include bacterial polysaccharides as well as carbohydrates of cancer cells or other non-self or target cell carbohydrates. In separale particular embodiments, the polysaccharides are the pneumococcal polysaccharides PnPs PnPsS, PnPs 6A and PnPs14. In various exemplary embodiments, the polysaccharides are from Pneumovax, Meningococcal, typhoid, cell surface glycolipids, glycoproteins, Haemophilus influenza Type b (Hib), RSV-F, staph, chlamydia, meningococcal type B, (Mening B), C. Difficile, Pseudomonas, Group A & B strep, enterotoxigenic Escherichia coii (ETEC), tuberculosis (IB), malaria toxin, Candida albicans, Shigella, Salmonella typhi, Salmonella paratyphi A, B, and C, C. botulinum, plague, Burkholcieria, and the like.
[0025] Exemplary PnPs polysaccharides include, but are not limited to, PnPsl , PnPs2, PnPs3, PnPs4, PnPsS, PnPs 6A, PnPsob, PnPs7F, PnPsS, PnPs9N, PnPs9V, PnPsl OA, PnPs1 1A, PnPs12F, PnPs14, PnPs15B, PnPs17F, PnPs18C, PnPs19F, PnPs19A, PnPs20, PnPs22F, PnPs23F, and PnPs33F of Streptococcus pneumoniae.
[0026] The amount of polysaccharide used in the fabrication of ihe virtual conjugate particle may be described with respect to the amount of the core particle. In particular, the amount of polysaccharide may be described with respect to the core particle: polysaccharide weight ratio. For example, the ratio of core particle to polysaccharide may be about 1 :0.001 (w:w), about 1 :0.002 (w:w), about 1 :0.003 (w:w), about 1 :0.004 (w:w), about 1 :0.005 (w:w), about 1 :0.01 (w:w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), or about 1 :0.05 (w:w). Thus, the polysaccharide used in the fabrication may be present in a range of about 1 :0.001 (w/w) to about 1 :0.05 (w/w). Typically, the polysaccharide is in a range of about 1 :0.01 (w:w) to about 1 :0.05 (w:w) (w/w). Often, the range is about 1 :0.01 (w:w) to about 1 :0.03 (w/w).
[0027] The amount of polysaccharide used in the fabrication of the virtual conjugate particle may also be expressed as a percentage of the core particle amount. For example, the polysaccharide may be present at about 0.1 % (w/w) of the core particle, about 0.2% (w/w) of the core particle, about 0.3% (w/w) of the core particle, about 0.4% (w/w) of the core particle, about 0.5% (w/w) of the core particle, about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, or about 5% (w/w) of the core particle. Thus, the polysaccharide used in the fabrication of the virtual conjugate particle may be present in a range of about 0.1 % to about 5% (w/w). Typically, the polysaccharide is in a range of aboul 0.1 % to about 5% (w/w). Often, the range is aboul 1 % to about 3% (w/w). Methods of producing particles to achieve these ratios are described in Example 9. in some cases, an active agent can also act as a carrier protein. Examples include, bui are ηοί limited to: pneumoiysoid protein (PLD), pneumococoai surface protein A (PspA), choline binding protein A (Cbp.A), PcsB-{proiein required for cell wall separation of group B streptococcus) and serine/threonine protein kinase (StkP) (inierceli), pneumococcal choline- binding protein A (PcpA), pneumococcal surface adhesion A (a substrate-binding lipoprotein of a manganese ABC transporter) (PsaA), serine/threonine protein kinase (StkP), pneumococcal hislidine triad proteins (PhtA B/D/E), and protein required for cell separation of group B streptococcus (PcsB).
[0028] Suitable nucleic acids for use in the invention include RNA, DNA, siRNA, shRNA, dsRNA, ssRNA, mi RNA, mRNA, rRNA, 1RNA, snR A, ssDNA, dsDNA, plasmid DNA, self- replication RNA, and antisense RNA.
Charged Biomoiecuie
[0029] In some aspects, the particles may contain a layer of charged biomolecuies that associate with the core particle structure and one or more particle components, including polysaccharides, linkers, and carrier proteins, by virtue of the electrostatic charge of the charged biomoiecuie. See, for example, Figure 1 , in certain aspects, an interaction may also comprise a covalent link, typically introduced using a cross-linker after the initial electrostatic-based interaction. In particular aspects, the charged biomoiecuie may be a caiionic biomoiecuie. The caiionic biomoiecuie may be selected from poiyeiecirolytes, charged polyaminoacids, charged polysaccharides, charged proteinaceous compounds, and charged peptides. Examples of positively charged biomolecuies include, but are not limited to, polyamino acids such as poiyiysine, polyhistidine, poiyornithine, polyciirulline, polyhydroxylysine, polyarginine, poiyhomoarginine, poiyaminotyrosine, histones, and protamines/protamine salts. Other suitable positively charged biomolecuies include, but are not limited to, polydiaminobulyric acid, poiyetbyieneimine, polypropyleneimine, polyamino(meth)acryl3te, polyaminosiyrene, poiyaminoethylene, poly(aminoetbyi)ethylene, polyaminoethylstyrene, diethyl amino ethyl cellulose, poiy-imino tyrosine, cholestyramine-resin, poly-imino acid, 1 ,5-dimethyi-1 ,5~ diazaundecametbylene poiymeihobromide (hexadimethrine bromide), chitosan, poiy(amidoamine) dendrimers, and combinations thereof Cationic biomoiecules of the present disclosure typically comprise one or more positively charged monomers. Examples of positively charged monomers include, but are not limited to, lysine, histidine, ornithine, hydroxyiysine, arginine, homoarginine, aminotyrosine, diaminobutyric acid, ethyieneimine, propyienimine, amino(meth}acryiaie, aminostyrene, aminoethylene, aminoethylethyiene, aminoethylstyrene, citruiline, diethyl amino ethyl glucose, imino tyrosine, (vinyibenzyi)trirnethyiammonium salts, imino acids, quaternary aikyi ammonium salts, amidoamines, glucosamine, and mixtures and derivatives thereof.
[0030] In other aspects, the charged biomoiecule may be a synthetic heteropolymer polypeptide containing only positively charged amino acids. For example, the charged biomoiecule may be a heteropolymer polypeptide of at least two of arginine, lysine, and histidine in a repeating unit, in some aspects, the repeating units are dimers or 1 Brimmers; for example, Arg~Lys dimers or Arg-Lys-His 16rimmers. In other aspects, the polypeptides may contain repeated combinations of dimers and 18rimmers. The arginine, lysine, and histidine residues rnay be arranged in any order. In yet other aspects, the heteropolymer polypeptide may contain non-positively charged amino acids. Typically, however, the heteropolymer polypeptide mostly contains positively- charged amino acids; for example, the polypeptide contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% positively-charged amino acids. Such polypeptides are readily synthesized by recombinant techniques.
[0031] Preferably the cationic biomoiecule is a protamine protein or a protamine protein salt. More preferably the cationic biomoiecule is a protamine salt. Examples of protamine salts include protamine sulfate, protamine chloride, protamine acetate, protamine propionate, protamine lactate, protamine formate, protamine nitrate, and protamine acetate. Most preferab y the cationic biomo!ecuie is protamine sulfate.
[0032] The amount of caiionic biomoiecuie used to fabricate the virtual conjugate particle may be described with respect to the amount of the core particle, in particular, the amount of cationic biomo!ecu!e may be described with respect to the core particle: cationic biomo!ecu!e weight ratio. For example, the ratio of core particle to cationic biomoiecuie may be about 1 :0.01 (w:w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), about 1 :0.05 (w:w), about 1 :0.06 (w:w), about 1 :0.07 (w:w), about 1 :0.08 (w:w), about 1 :0.09 (w:w), or about 1 :0.1 (w:w). Thus, the polysaccharide may be present in a range of about 1 :0.01 (w/w) to about 1 :0.1 (w/w). Typically, the polysaccharide is in a range of about 1 :0.03 (w:w) to about 1 :0.08 (w:w) (w/w). Often, the range is about 1 :0.04 (w:w) to about 1 :0.08 (w/w).
[0033] The amount of cationic biomoiecuie used to fabricate the virtual conjugate particle may also be expressed as a percentage of the core particle amount. For example, the cationic biomoiecuie may be present at about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, about 5% (w/w) of the core particle, about 6% (w/w) of the core particle, about 7% (w/w) of the core particle, about 8% (w/w) of the core particle, about 9% (w/w) of the core particle, or about 10% (w/w) of the core particle. Thus, in aspects, the cationic biomoiecuie may be present in a range of about 1 % to about 10% (w/w). Typically, the cationic biomoiecuie is in a range of about 3% to about 8% (w/w). Often, the range is about 4% to about 6% (w/w). Methods of producing particles to achieve these ratios are described in Example 9.
Cross-Linker
[0034] in embodiments, the particles may be modified by chemically cross-linking the charged protein, entangling the antigenic substance near the surface of the particle. In some embodiments, the proteins can be chemically cross-linked either through the nitrogen functionality of N-ierminus or lysine residues or the thiol functionality of cysteine residues using an appropriate difunctional cross-linker. In other embodiments, proteins can be chemically cross-linked through carboxyiates to primary amines.
[0035] Suitable cross-linkers for use in the invention include, but are not limited to 1-ethyi-3-(3- dimethylaminopropyi) carbodiimide (EDC), giutaraidehyde (GA), bismaleimidohexane (BMH), dimethyl suberirnidate (DMS), dimethyl pime!imidaie (D P), 4-(4,6~dimethoxy-1 ,3l5-trianzi-2~yl)~ 4-methylmorphoiiniurn chloride, 1-cyano-4-dimetbyiaminopyndinium tetrafiuoroborate (CDAP), N-hydroxysuccinimide esters (NHS), dithiobis(succinimidyi propionate(DTSP), 4-(4,6~dimethyl- 1 ,3,5-triazin-2-yi) (DMTMM) Des Martin Periodinane, Tempo/ aGiG4, 2-iminoihioione, adipic dihydrazide (ADM), aminoxyaletate, carbonyl diimidazole, disuccinimidyl carbonate, and combinations thereof.
[0036] in some embodiments, the N-hydroxysuccinimide ester is selected from the group consisting of dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), succinimidyl 3~(2~pyhdydithio) propionate (SPDP), succinimydyl 4~[N- maieimidomethyl] cyciohexane-1-carboxyiate (SMCC), sulfosuccinimidyi-4-[N-rnaleimidomethyi] cyclehexane-1-carboxylate (suifo-S CC), and sulfosuccinimidyl 8-(3'-[2- pyridydithiojpropionamido) hexanoate (suifo-LC-SPDP).
[0037] Preferably the cross-linker is 1-ethyl-3-(3-dimethyiarninopropyi) carbodiimide (EDC), giutaraidehyde (GA), aclipic dihydrazide (ADH), or bismaleimidohexane (BMH). More preferably the cross-linker is 1-ethyl~3~(3-dimethyiaminopropyi) carbodiimide (EDC) or giutaraidehyde (GA). Most preferably the cross-linker is giutaraidehyde (GA).
[0038] In some embodiments, the antigenic polysaccharide is PnPsI , PnPs 6A, or typhoid, and the cross-linker is EDC. in other embodiments, the antigenic polysaccharide is PnPsI , PnPs5, or PnPs14, and the cross-linker is glutaraidehyde (GA). In other embodiments, the antigenic polysaccharide is PnPs 6A and the cross-linker is ADH.
Carrier Protein
[0039] The pharmaceutical composition particle of the present invention may also include a carrier protein. Suitable carrier proteins for use in the invention include, but are ηοί limited to CRM tetanus toxoid, diphtheria toxoid, hemagglutinin, meningococcal surface proteins, pseudomonas aeruginosa endotoxin A (r-EPA), T-cei! protein antigens, ovalbumin, and capsular proleins. CR 97 is an example of a non-toxin, mutant protein derived from diphtheria ioxin (DT) and that has long been recognized to be a non-toxic protein and is commonly used as a carrier for conjugate vaccines. Based on its non-toxic feature, this protein has been utilized for various purposes, including as an inhibitor of beparin-binding EGF-like growth factor (HB-EGF) and as an immunological adjuvant for vaccination. Other suitable carrier proteins include, but are ηοί limited ίο S. Aureus; aggiuUnin-like sequence (Ais), including Ais3, which promotes adhesion to the endotheiiial cells {also for an antigen for Candida) (Novadigm), dumpling Factor A (ClfA) (Wyetb/Pfizer), clumping factor 8 (CifB), iron surface determinant B (isdB), Can-FnBP fusion protein, Poly-M-acetyl glucosamine; meningitis B; GNA 1870, also named factor H- binding protein (fHbp) or rLP-2086 (Novartis); Group B Strep: Protein Antigens c, R and X.ln some cases, an active agent can also act as a carrier protein. Examples include, but are not limited to: pneumolysoid protein (PLD), pneumococcal surface protein A. (PspA), choline binding protein A (CbpA), Pcs8-{protein required for cell wail separation of group B streptococcus) and serine/threonine protein kinase (StkP) (Intercell), pneumococcal choline-binding protein A (Pc-pA), pneumococcal surface adhesion A {a substrate-binding lipoprotein of a manganese ABC transporter) (PsaA), serine/threonine protein kinase (StkP), pneumococcal bistidine triad proteins (PbtA/B/D/E), and protein required for ceil separation of group B streptococcus (PcsB), [0040] Preferably the carrier protein is pneumolysold protein (PLD), CRM-|97, pneumococcal surface protein A (PspA), or diphtheria toxoid. More preferably the carrier protein is Pneurno ysoid protein (PLD) or CR 97. Most preferably the carrier protein is pneurnolysoid protein (PLD).
[0041] The amount of carrier protein used to fabricate the virtual conjugate particle may be described with respect to the amount of the core particle. In particular, the amount of carrier protein may be described with respect to the core particle: carrier protein weight ratio. For example, the ratio of core particle to carrier protein may be about 1 :0.01 ( :w), about 1 :0.02 (w:w), about 1 :0.03 (w:w), about 1 :0.04 (w:w), about 1 :0.05 (w:w), about 1 :0.06 (w:w), about 1 :0,07 (w:w), about 1 :0.08 (w:w), about 1 :0.09 (w:w), or about 1 :0.1 (w:w). Thus, the carrier protein may be present in a range of about 1 :0.01 (w w) to about 1 :0.1 (w/w). Typically, the polysaccharide is in a range of about 1 :0.03 (w:w) to about 1 :0.08 (w:w) (w/w). Often, the range is about 1 :0.04 (w:w) to about 1 :0.08 (w/w).
[0042] The amount of carrier protein used to fabricate the virtual conjugate particle may also be expressed as a percentage of the core particle amount. For example, the carrier protein may be present at about 1 % (w/w) of the core particle, about 2% (w/w) of the core particle, about 3% (w/w) of the core particle, about 4% (w/w) of the core particle, about 5% (w/w) of the core particle, about 6% (w/w) of the core particle, about 7% (w/w) of the core particle, about 8% (w/w) of the core particle, about 9% (w/w) of the core particle, or about 10% (w/w) of the core particle. Thus, in aspects, the carrier protein may be present in a range of about 1 % to about 10% (w/w). Typically, the carrier protein is in a range of about 3% to about 8% (w/w). Often, the range is about 4% to about 6% (w/w). Methods of producing particles to achieve these ratios are described in Example 9
[0043] The amount of polysaccharide and carrier protein in the virtual conjugate particles may also be described with respect to the ratio between these two components. For example, the ratio of polysaccharide to carrier protein may be in a range between about 0.001 and about 0.5, between about 0.01 and about 0.5, between about 0.01 and about 0.4, and between about 0,02 and about 0.4. In preferred aspects, the ratio is between about 0.02 and about 0.35.
Additional Components
[0044] The particle can include other agents, excipients or stabilizers. For example, to increase stability or decrease non-specific uptake by increasing the negative zeta potential of nanopartieles, certain negatively charged components may be added. Such negatively charged components include, but are not limited to bile salts of bile acids consisting of glycocholic acid, cholic acid, chenodeoxycholic acid, taurochoiic acid, giycochenodeoxychoiic acid, taurochenodeoxychoiic acid, iitochoiic acid, ursodeoxycholic acid, dehydrocholic acid and others: phospholipids including lecithin (egg yolk) based phospholipids which include the following phosphatidylcholines: paimitoyioleaylphosphaticiylchoifne, paimitoyilinoieoyiphosphatidyichoiine, stearoyilinoieoyiphosphatidyicholine stearoyioieoyiphosphatidylehoiine, stearoyiarachidoylphosphatidyicholine, and dipalmitoyiphosphatidyicholine. Other phospholipids including L-o dimyristoylpbospbatidyicholine (DMPC), dioieoylphosphatidyicholine (DOPC), distearoylphosphatidylchoiine (DSPC), hycirogenateci soy phosphatidylcholine (HSPC), and other related compounds. Negatively charged surfactants or emulsifiers are also suitable as additives, for example, sodium cholesieryi sulfate and the like. Similarly, the positive zeta potential of the core particles can be altered by adding positively charged components. Such positively charged components incksde, but are not limited to cationic lipids such as 1 ,2-dioieoyl- 3-trimethy!ammoniumpropane chloride (DOTAP), and 3β-[Ν-(Ν\ N'-dirnethyiaminoetbane)- carbamoyl] cholesterol hydrochloride (DC-Ghol). Routes of Administration
[0045] The conjugate particles have also found use for delivery of a pharmaceutical composition, such as a vaccine, that may be administered via various routes including orally, derrnally, inhalation, intravenously, intramuscularly, subcutaneousiy, intra-artehaily, intraperitoneally, intranasally, inlrapulmonary, intraoculariy, intravasculahy, intralhecaiiy, or intratracheal!'/.
DETAILED DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 depicts one embodiment of a pharmaceutical composition particle 100 of the invention. The pharmaceutical composition particle 100 comprises a base particle 101 with a cationic biomoiecule 102 associated with the surface of the core particle. Associated with the cationic biomoiecule 102 are a polysaccharide 103 and a carrier protein 104. A cross-linker 105 is associated with the polysaccharide 103, carrier protein 104, and cationic biomoiecule 102.
[0047] In one embodiment, the core particle 101 is poly(iactide-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide 103 is pneumococcal polysaccharide PnPs 5, carrier protein 104 is PLD, and cross-linker 105 is giutaraidehyde, in another embodiment, the core particle 101 is poly(lactide-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide is pneumococcal polysaccharide PnPs 14, carrier protein 104 is PLD, and cross-linker 105 is giutaraidehyde. In another embodiment, the core particle 101 is poly(!actide-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide is pneumococcal polysaccharide PnPs 1 , carrier protein 104 is PLD, and cross- linker 105 is 1 -ethyl-3-[3-dimethylaminopropyi]carbodiimide hydrochloride (EDC). In another embodiment, the core particle 101 is poly(lacticie-co-giycolide) polymer, cationic biomoiecule 102 is protamine sulfate, polysaccharide 103 is pneumococcal polysaccharide PnPs 14, carrier protein 104 is ovalbumin (OVA), and cross-linker 105 is glutaraldehyde. in another embodiment, the core particle 101 is poly(lacUde-co-giycolide) polymer, cationic biomolecule
102 is protamine sulfate, polysaccharide 103 is pneumococcal polysaccharide PnPs 14, carrier protein 104 is CR ^T, and cross-linker 105 is glutaraldehyde.
[0048] FIG. 2 depicts a second embodiment of a virtual conjugate particle 200 of the invention. The virtual conjugate particle 200 comprises a core particle 101 with a carrier protein 104 and polysaccharide 103 associated with the surface of the particle. A cross-linker 105 is associated with the polysaccharide 103 and carrier protein 104.
[0049] In one embodiment, the core particle 101 is poly(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide 103 is pneumococcal polysaccharide PnPs 5, carrier protein 104 is PLD, and cross-linker 105 is glutaraldehyde. In another embodiment, the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide
103 is pneumococcal polysaccharide PnPsl , carrier protein 104 is PLD, and cross-linker 105 is 1-ethyl-3-[3-dimethylaminopropyl]carbod{imide hydrochloride (EDC). In another embodiment, the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide 103 is pneumococcal polysaccharide PnPs 1 , carrier protein 104 is PLD, and cross-linker 105 is bismaleimidohexane (B H). In another embodiment, the core particle 101 is poiy(lactide-co-glycolide) polymer blended with DC-cholesterol, polysaccharide 103 is Vi polysaccharide, carrier protein 104 is CRM5g- , and cross-linker is glutaraldehyde.
[0050] FIG. 3 depicts a third embodiment of a virtual conjugate particle 300 of the invention. The virtual conjugate particle 300 comprises a core particle 101 with a cationic biomolecule 102 associated with the surface of the core particle. Associated with the cationic biomolecule 102 is a nucleic acid 106.
[0051] In one embodiment, the core particle 101 is poly(iactide-co-giycolide) polymer, cationic biomolecule 102 is protamine sulfate, and nucleic acid 106 is influenza HA R A. in another embodiment, ihe core particle 101 is poly(iactide~co-giycolide) polymer, cationic biomolecu!e 102 is protamine sulfate, arid nucleic acid 108 is RSV-F RNA,
[0052] FIG. 4 depicts a fourth embodiment of a virtual conjugate particle 400 of the invention. The virtual conjugate particle 400 comprises a core particle 101 with a nucleic acid 106 and a cationic biomolecule 102 associated with the surface of Ihe particle.
[0053] In one embodiment, the core particle 101 is poly{lacticle-co-glycolide) polymer blended with DC-cholesterol, nucleic acid is influenza HA RNA, and cationic biomolecule 102 is protamine sulfate.
[0054] One aspect of the present invention is the surface coating of the core particles with a charged species. Additional components can then be adsorbed to the surface of the core particles after application of the surface coating. These components can be charged or neutral moieties including but not limited to proteins (OVA, CRM, TT, PLD), polysaccharides (Vi PnPs, pneumococcal PnPs strains, synthetic PnPs), and nucleic acids. In some embodiments, core particles with surface adsorbed components can be treated with a stoichiometric excess in relation to the amount of protein being added of a cross-linking agent,
[0055] FIG. 5 depicts a "one pot" process for producing the virtual conjugate particle 100 of the invention. A plurality of core particles 101 are suspended in water or buffer solution. A cationic biomolecule 102 is added to the suspension and the combination is agitated with rocking at room temperature. After a period of time, for example approximately ten minutes, polysaccharide 103 is added to the suspension and the combination is agitated with rocking at room temperature. After a period of time, for example approximately ten minutes, carrier protein
104 is added to the suspension and the combination is agitated with rocking at room temperature. Next, after approximately ten minutes, additional cationic biomolecule 102 and cross-linker 105 are added to the suspension. The suspension is agitated with rocking at room temperature for a period of time, for example approximately one hour. The cross-linker 105 is neutralized, if applicable, and the resulting virtual conjugate particles are washed and recovered through centrifugaiion.
[0056] In one embodiment of the "one pot" method of making the pharmaceutical composition, the overall volume of the reaction is maintained at 1 mL 2 mg of poly (lactic acid-co-glycoiic acid) core particles 101 are suspended in water or a buffer solution. 58.18 pg of protamine sulfate (in solution), a cationic biomoiecule 102, is added to the suspension. After approximately ten minutes of agitation at room temperature, 44.94 pg of pneumococcal polysaccharide PnPsl (in solution), a polysaccharide 103, is added to the suspension without removing the previous solution. After approximately ten minutes of agitation at room temperature, 89.888 pg of PLD protein (in solution), a carrier protein 104, is added to the suspension without removing the previous solution. After approximately ten minutes of agitation at room temperature, additional 56.18 pg of protamine sulfate (in solution) and glutaraldehyde, a cross-linker 105, is added to 0,70% based on the total volume (1 mL) of the reaction, both without removing the previous solution. The sample is agitated for approximately one hour at room temperature. After approximately one hour the was quenched using 35 mM sodium cyanoborohydride. The pharmaceutical composition is recovered through centrifugaiion and washing of the particles.
[0057] FIG. 6 depicts a second process for producing the virtual conjugate particle 100 of the invention. A plurality of core particles 101 are suspended in water or buffer solution. The cationic biomoiecule 102 is added to the suspension and incubated with the core particles 101 for a period of time, for example overnight, with rocking. The resulting coated core particles are centrifuged, washed and re-suspended in water or buffer solution. Polysaccharide 103, carrier protein 104, and cross-linker 105 are added to the suspension which is then incubated for a period of time, for example approximately one hour, at room temperature with rocking. The cross-linker 105 is neutralized, if applicable, and the resulting virtual conjugate particle 100 is Ihen centrifuged, washed and recovered from the suspension through centrifugation.
[0058] In one embodiment, a plurality of poly (lactic acid-co-glycoiic acid) core particles 101 are suspended in water or a buffer solution. A 5% protamine sulfate solution, a cationic biomolecuie 102, are added and the suspension is rocked at room temperature overnight. The unbound protamine is then removed using centrifugation and water washing. Pneumococcal polysaccharide PnPsS or PnPs 14 (2% solution), carrier protein PLD (4% solution), and glutaraldehyde (1 % solution) are then added and the suspension is rocked for approximately one hour at room temperature. 35m sodium cyanoborohydride (NaCNBH3) is added to quench the reaction. The pharmaceutical composition is recovered through centrifugation and washing of the particles.
[0059] FIG. 7 depicts a third process for producing the virtual conjugate particle 100 of the invention, A cationic biomolecuie 102 and carrier protein 104 are combined in a solution and incubated for a period of time, for example approximately one hour. This solution is then added to a suspension of core particles 101 and mixed for a period of time, for example approximately one hour, at room temperature with agitation. The resulting particles are centrifuged, washed and re-suspended in water or buffer solution. Polysaccharide 103 and cross-linker 105 are added to the suspension and the mixture is incubated for a period of time, for example overnight, at room temperature with rocking. The cross-linker is neutralized, if applicable, and the resulting virtual conjugate particles are centrifuged, washed and recovered from the suspension through centrifugation,
[0060] In one embodiment, a 5% protamine sulfate solution and a 4% PLD carrier protein solution are combined in a 0.5 mL tube and allowed to incubate at room temperature for one hour. The protamine/protein mixture are added to a plurality of poly (lactic acid-co-giycolic acid) core particles. The suspension is rocked in the tube at room temperature for one hour. Unbound protamine and/or protein are removed using centrifugation and water washing.
Pneumococcal polysaccharide PnPsl (2% solution) and 1~ethyl-3-[3~ dimeihylaminoprapyl carbodiirnide hydrochloride (EDC) are added. The pharmaceutical composition is recovered through centrifugation and washing of the particles.
Use of the Particle in a Pharmaceutical Composition
[0061] The pharmaceutical compositions of the present invention may be used for both prophylactic and therapeutic purposes.
[0062] The particles and compositions containing the particies may be administered to any subject in need. Typically, the subject is a mammalian subject. Often, the mammalian subject is a human, in other aspects, the particle and composition may be used for veterinary purposes and administered to an animal may it be a cat, dog or other animal
[0063] One particular use for the drug delivery system of the present invention is as a vaccine. Accordingly, the invention provides for the use of a vaccine for the treatment of human patients. The invention further provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient, in particular, the invention provides a method of treating viral, bacterial, or parasitic infections or cancer which comprises administering an effective amount of a drug delivery composition of the present invention to a patient.
[0064] An appropriate vehicle for administration of the drug delivery composition is a solution or suspension containing the particles. The particles can also be part of a lyophilized composition prior to administration. The lyophilized composition can be reconsititued into a solution or suspension prior to administration. The particles may be delivered through various routes including subcutaneously, intravenously, intraderrnaliy, or intramuscularly in a solution or suspension. For example, the pharmaceutical composition may contain the target amount of antigenic material in an aqueous solution of mannitoi and polyvinyl alcohol, containing for example (wt%) 5% mannilol and 0.1 % polyvinyl alcohol,
[0065] The virtual conjugate particles may contain a single antigen or multiple antigens of interest. Virtual conjugate particles may be blended to produce a pharmaceutical composition with a multitude of antigens. For example, three monovalent virtual conjugate particle compositions can be blended to form a pharmaceutical composition, in this example, three separate monovalent particle suspensions of known particle concentrations, in mg/mL, are combined into a trivalent particle suspension by transferring a calculated volume of each particle suspension into one vial. The calculated volume contains a known mass of particles based on the particle concentration of each suspension. The particle suspension can be from a freshly made batch of virtual conjugate particles or from a reconstituted sample of lyophilized virtual conjugate particles.
[0066] The pharmaceutical composition dose can be calculated based on delivery of a specified mass of virtual conjugate particles, for example 100 pg. in that case, a specific mass of each monovalent virtual conjugate particle composition is measured by weight or volume. In other instances, the dose is based on delivering a specified mass of antigen. For example, 2,2 pg polysaccharide or 10 pg protein, in this case the loading of the antigen on the virtual conjugate particle must be known.
[0067] A series of examples will be further described. The following examples are not intended to be limiting but only exemplary of specific embodiments of the present invention.
EXAMPLES
[0068] Sources of the components to produce the virtual conjugate particles are detailed in the table below, Component Abbreviation Function
Poly(lactide-co-glycolide) polymer PLGA Core particle
DC-cholesterol DC-Choi Cationicbiomolecule
Pneumococcal polysaccharide PnPsl Polysaccharide
PnPsl
Pneumococcal polysaccharide PnPs5 Polysaccharide
PnPs5
Pneumococcal polysaccharide PnPs 6A Polysaccharide
PnPs 6A
Pneumococcal polysaccharide PnPs14 Polysaccharide
PnPs14
Pneumococcal polysaccharide PnPs 19A Polysaccharide
PnPs 19A
Vi polysaccharide Vi Polysaccharide
self-replicating RNA encoding for HA-RNA Nucleic acid
HA (hemagglutinin) RNA
self-replicating RNA encoding for RSV-F-RNA Nucleic acid
RSV-F (respiratory syncytial virus
protein F) RNA
Ovalbumin OVA Protein
Diptheria toxin mutant, cross CRM197 Protein
reacting material
Pneumolysoid protein PLD Protein
Protamine sulfate PA Cationic biomolecule
Glutaraldehyde GA Cross-linker
1-ethyl-3-[3- EDC Cross-linker
dimethylaminopropyl]carbodiimide
hydrochloride
Bismaleimidohexane BMH Cross-linker
[0069] ΑΠ PnPs are from Ihe Serum Institute of India (SH) and are native unless otherwise noted.
Example 1
Production of 80 by 80 nm by 320 nm PLGA Particle [0070] Particles were manufactured utilizing PRINT© Technology and poly(iaeiide-co-glycolide) polymer. These methods have previously been described in, for example U.S. Pat. os. 8,518,316; 8,444,907: 8,420, 124; 8,268,446; 8,263,129; 8,158,728; 8,128,393; 7,976,759; U.S. Pat. Application Publications Nos. 2013-0249138, 2013-0241 107, 2013-0228950, 2013- 0202729, 2013-0011618, 2013-0256354, 2012-0 89728, 2010-0003291 , 2009-0 65320, 2008- 0131692; and pending applications 13/852,683 filed March 28, 2013 and 13/950,447 filed July 25, 2013.
Example 2
Production of 200 nm by 200 nm by 200 nm PLGA Particle
[0071] Example 1 was repeated using a FLUOROCUR'"'1 Moid with 200 nrn by 200 nm by 200 nm cubical cavities.
Example 3
Production of 80 nm by 80 nm by 320 nm PLGA/DC-Cholesterol Particle
[0072] Example 1 was repeated using poly(lactide-co-glycolide) polymer blended with 10% by weight DC-cholesterol.
Example 4
Production of 80 nm by 80 nm by 320 nm PLGA/DMAEMA Particle
[0073] Example 1 was repeated using poly(lactide-co-glycolide) polymer blended with 3% by weight dimethyiaminoethyi methacrylate (DMAEMA).
Example 5
Production of PLGA/Protamine Glutaraldehyde Cross-linked Particles [0074] A mass of particles fabricated by any of Example 1 , Example 2 or Example 4 were aliquoied into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL A 5% {5 mg/mL in water) protamine sulfate solution was added and the suspension was rocked at room temperature overnight. To wash away unbound protamine, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 μΐ water until no particle chunks were present. The volume was increased to 1 ml to continue to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in water.
[0075] Polysaccharide, protein, and giutaraidehyde (cross-linker) were added to the particle suspension using aqueous stock solutions. The polypropylene tube was placed on a rocker for one hour at room temperature. 35m sodium cyanoborohyd ide (NaCNBH3) was added to quench the reaction. The tube was rocked one hour at room temperature. To wash away unbound components, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 pL water until no particle chunks were present. The volume was increased to 1 mL water to continue to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in 0.1 % polyvinyl alcohol/5% mannitoi.
Example 6
Production of PLGA/Protamine EDC Cross-linked Particles
[0076] Protamine and protein were combined in a 0.5 mL tube and allowed to incubate at room temperature for one hour. The protamine/protein mixture was added to an aliquot of the particles of any of Example 1 or Example 2 and water was added to achieve a targeted concentration, for example 2 mg/mL. The suspension was rocked in the tube at room temperature for one hour. To wash away unbound protamine and/or protein, the suspension was centrifuged at 12.500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 μΐ water until no particle chunks were present. The volume was increased to 1 mL to continue to wash the particles. The suspension was centrifuged a second lime at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re- suspended to a targeted concentration, for example 2 mg/mL, in water.
[0077] Polysaccharide and 1-ethyi~3-[3-dimethyiaminopropyi]carbodiimide hydrochloride (EDC, cross-linker) were added to the particle suspension as stock solutions and the polypropylene tube was placed on a rocker at room temperature overnight. To wash away unbound components, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 μΐ water until no particle chunks were present. The volume was increased to 1 ml to continue to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 mg/mL, in 0.1 % polyvinyl alcohol/5% mannitol.
Example 7
Production of PLGA/DC-Chol Glutaraldehyde Cross-linked Particles
[0078] Polysaccharide, protein, and glutaraldehyde (cross-linker) were added to a particle suspension of Example 3 using aqueous stock solutions. The polypropylene tube was placed on a rocker for one hour at room temperature. 35 mM sodium cyanoborohydride (NaCNBH3) was added to quench the reaction. The tube was rocked one hour at room temperature. To wash away unbound components, the suspension was cenirifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 pL water until no particle chunks were present. The volume was increased to 1 ml to continue to wash the particies. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended to a targeted concentration, for example 2 rog/mL, in 0.1 % polyvinyl alcohol/5% mannitoi.
Example 8
Production of PLGA DC-Chol EDC Cross-linked Particles
[0079] Polysaccharide, protein, and 1-ethyi~3-[3-dimethyiaminopropyi]carbodiimide hydrochloride (EDC, cross-linker) were added to an aliquot of the particles of Example 3 and water was added to achieve a targeted particle concentration, for example 2 mg/rnL and the polypropylene tube was placed on a rocker at room temperature overnight. To wash away unbound components, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 pi water until no particle chunks were present. The volume was increased to 1 mL to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re-suspended a targeted concentration, for example 2 mg/rnL, in 0.1 % polyvinyl alcohol/5% mannitoi.
Example 9
Sample Calculations to Produce Virtual Conjugate Particles
[0080] A series of stock: solutions were used to produce the virtual conjugate particies of
Examples 5 through Example 8. The various additions were calculated as described below. The core particle is a suspension while the protamine protein, polysaccharide, protein, and cross-linker are solutions in fabrication. Consequently, in fabrication, unbound protamine protein, polysaccharide, protein, and cross-linker are washed away. Those percentages or masses are noted as fabrication percentages or fabrication masses respectively. The total fabrication mass is noted as such as the value is comprised of the masses of the protamine protein, polysaccharide, protein, and cross-linker during fabrication.
The total fabrication mass of the virtual conjugate particles was equal to the sum of the core particle mass plus the protamine protein fabrication mass plus the polysaccharide fabrication mass plus the protein fabrication mass. Expressed mathematically:
Total fabrication mass of virtual conjugate particles (mg) = core particle (rng) + fabrication protamine (mg) + fabrication polysaccharide (mg) ÷ fabrication protein (mg)
[0081] Alternatively, the total fabrication mass of the virtual conjugate particles was also calculated based on the targeted component percentages for the virtual conjugate particle. Using this method of calculation, the total fabrication mass of the virtual conjugate particles was equal to the core particle mass divided by the core particle mass percent, where the core particle mass percent was equal lo one minus Ihe prolamine protein target fabrication percenl minus the polysaccharide target fab ication percent minus the protein target fabrication percent. Expressed mathematically:
Total fabrication mass of virtual conjugate particles (mg) = core particle (mg) / (1 - fabrication % protamine - fab ication % polysaccharide - fabrication % protein), where fabrication % protamine, fabrication % polysaccharide, and fabrication % protein are the targeted fabrication percentages, expressed as a decimal, in Ihe virtual conjugate particle.
[0082] Once the total fabrication mass of the virtual conjugate particles was calculated, the target fabrication mass of each component was calculated. For each component, the total fabrication mass of the virtual conjugate particles was multiplied by the respective component target fabrication percentage wherein Ihe target fabrication percentage was expressed as a decimal. Expressed mathematically:
Protamine target fabrication mass (mg) = (fabrication % protamine) * (total fabrication mass of virtual conjugate particles (mg))
Polysaccharide target fabrication mass (mg) = (fabrication % polysaccharide) * (total fabrication mass of virtual conjugate particles (mg))
Protein target fabrication mass (mg) = (fabrication % protein) * (total fabrication mass of virtual conjugate particles (mg))
[0083] Once the target fabrication mass additions were calculated for each component, the target volume for each component for the stock solution was calculated. To calculate this value the component target fabrication mass, in mg, was divided by the concentration of the stock- solution, in mg/mL Expressed mathematically:
Protamine target volume (ml.) = Protamine target fabrication mass (mg) / (concentration protamine stock solution (mg/mL)), to convert to μΙ_, the result was multiplied by 1000.
Polysaccharide target volume (ml.) = Polysaccharide target fabrication mass (mg) /
(concentration polysaccharide stock solution (mg/mL)), to convert to pL, the result was multiplied by 1000.
Protein target volume (mL) = Protein target fabrication mass (mg) / (concentration protein stock solution (mg/mL)), to convert to pL, the result was multiplied by 1000.
[0084] For example, 1 rng of core particles was used to produce virtual conjugate particles with targeted component fabrication percentages of 5% protamine, 2% polysaccharide, and 4% protein.
[0085] To calculate the total fabrication mass of the virtual conjugate particles, the mass of the core particles was divided by the core particle mass percent, wherein the core particle mass percent was equal to one minus the protamine target fabrication percent minus the polysaccharide target fabrication percent minus the protein target fabrication percent. Mathematically;
Total fabrication mass of virtual conjugate particles (mg) = 1 mg / (1 - 0.05 - 0.02 - 0.04) = 1 mg / 0.89 = 1 .1236 mg
To calculate the target fabrication mass for each component, the total fabrication mass of virtual conjugate particles was multiplied by the target fabrication percent for each respective component. Mathematically:
Protamine target fabrication mass (mg) = 0.05 * (1 .1236 mg) = 0.0562 mg
Polysaccharide target fabrication mass (mg) = 0.02 * (1 .1236 mg) = 0.0225 mg
Protein target fabrication mass (mg) = 0.04 * (1 .1236 mg) = 0.0449 mg
[0086] To calculate the target volume for each component, the target fabrication mass of the component, in mg, was divided by the stock solution concentration, in mg/mL. The result was multiplied by 1000 to obtain μΐ, Mathematically:
Protamine target volume (ml.) from a 5 mg/mL stock solution: 0.0562 mg / (5.00 mg/mL) = 0.01 124 mL = 1 1 .24 pL
Polysaccharide target volume (mL) from a 5.33 mg/mL stock solution: 0.0225 mg / (5.33 mg/mL) = 0.00422 mL = 4.22 pL
Protein target volume (mL) from a 3.50 mg/mL stock solution: 0.0449 mg / (3.50 mg/mL) = 0.01282 mL = 12.82 pL
Cross-linking Reaction: Giutaraidehyde in Example 5 and Example 7
[0087] All surface reactions were conducted such that the core particle target concentration was approximately 2 mg/mL. Referring back to the previous example of 1 mg of core particles, the target reaction volume was 0.5 ml (1 mg/0.5 ml = 2 mg/ mL), Of that 0.5 ml, 1 1 .24 pL + 4,22 pL + 12.82 pL were stock solutions, 250 pL was a 1 .4% giutaraidehyde stock solution and the ba ance was water (221 ,71 pL). The net glutaraldehyde concentration was then 0.7% based on the 50% dilution.
[0088] To neutralize the glutaraldehyde, sodium cyanoborohydnde was added at a two times molar excess based on the glutaraldehyde present in the reaction.
Cross-linking Reaction: EDC in Example 8 and Example 8
[0089] When using EDC as a cross-linker, the target amount of EDC added was relative to the target fabrication mass of the carrier protein added. For particles of Example 6, the target amounl of EDC added was ten times the target fabrication mass of carrier protein added. Mathematically:
EDC target mass (mg) = 0.0449 mg * 10 = 0.4490 mg
Cross-linking Reaction: BMH
[0090] When using BMH as a cross-linker, the target amount of BMH added was relative to the target fabrication mass of the carrier protein being added. The target amount of BMH added was two times the target fabrication mass of the carrier protein added. Mathematically:
BMH target mass (mg) = 0.0449 mg * 2 = 0.0900 mg Example 10
Production of PLGA/DC-Chol. Particles with self-replicating RNA encoding for HA-RNA
[0091] Particles of Example 3 were washed three times in nuclease-free water. The particles were re-suspended in water to a concentration of 8.3 mg/mL 602 pi particles were combined with 800.2 μΐ water, and 97.8 μί_ of a 1.1 mg/mL solution of HA-RNA. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 12,000 X g for twenty minutes at 4°C. The resulting pellet was re-suspended in 2 mL of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 12,000 X g for twenty minuies at 4°C. The resulting pellet was re-suspended in 365.5 pL water and 134.4 pi of a 2% prolamine sulfate solution were added. The suspension was rocked for one hour at room temperature. The particles were washed again as described above. The final sample was diluted to 5.37 mg/mL using an aqueous (wt%) 0.2% polyvinyl alcohol/10% mannito! solution.
Example 11
Production of PLGA/Protamine Particles with self-replicating RNA encoding for HA-RNA
[0092] 125 pL particles of Example 2 were combined with 1 1 15 pL 0.1 % polyvinyl alcohol and 67,2 pi of a 2% protamine sulfate solution. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 10,000 X g for twenty minutes at 4"C and the supernatant was decanted off. The resulting pellet was re- suspended in 2 ml of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 10,000 X g for twenty minutes at 4°C and the supernatant was decanted off. The pellet was re- suspended in 201 pL water and 97.8 pL of a 1.1 mg/mL solution of HA-RNA was added. The suspension was rocked at room temperature for one hour. The sample was diluted to 1 mL using a 0.2% polyvinyl alcohol/10% mannito! solution to a final concentration of 5.48mg/mL
Production of PLGA/Protamine Particles with self-replicating RNA encoding for RSV-F-RNA
[0093] 125 pL particles of Example 2 were combined with 1 1 15 pL 0.1 % polyvinyl alcohol and 87.2 pL of a 2% protamine sulfate solution. The suspension was rocked at room temperature for thirty minutes. To wash the particles, the suspension was centrifuged at 10,000 X g for twenty minutes at 4°C. The resulting pellet was re-suspended in 2 mL of 0.1 % polyvinyl alcohol. The suspension was centrifuged again at 10,000 X g for twenty minutes at 4°C. The pellet was re-suspended in 225 pL water and 51 pL of a 2.1 mg/mL solution of RSV-F-RNA was added. The suspension was rocked at room temperature for one hour. The sample was diluted to 1 mL using 0.2% polyvinyl alcohol/10% mannito! to a final concentration of 5.48 mg/mL. Example 12
Animal Studies and Controls
[0094] A series of animai studies were conducted to determine the efficacy of various vaccine formulations comprised of virtual conjugate particles. Both BALB/c mice and New Zealand While rabbits were utilized in the studies. Six animals were inoculated for each sample unless otherwise noted. Inoculation schedule and route of administration is noted in each exampie. Animals in studies involving Streptococcus Pneumoniae polysaccharides received boost injections al T = four weeks (Boost 1 ) and T = eight weeks (Boost 2). Serum was collected for analysis at T = 0 {Prime), Post Prime, Post Boost 1 and T = 10 weeks (Post Boost 2). Production of the virtual conjugate particles is described in each respective animal study below.
[0095] Commercial vaccines were used as controls. For studies incorporating Streptococcus pneumoniae polysaccharides, PREVNAR 13® Pneumococcal 13-vaient Conjugate Vaccine (Diptheria CR i97 Protein) (Pfizer, inc., Philadelphia, PA) was used as a control. For studies incorporating Vi polysaccharide, TYPHi Vi© Typhoid Vi Polysaccharide Vaccine was used as the control.
[0096] PREVNAR 13© is a commercially available pneumococcal conjugate vaccine manufactured by Wyeth Pharmaceuticals, inc. and marketed by Pfizer, inc. The vaccine contains purified capsular polysaccharide from thirteen of the Streptococcus pneumoniae serotypes; PnPsl , PnPs3, PnPs4, PnPsS, PnPs6A, PnPs6B, PnPs7F, PnPs9V, PnPs14, PnPs18C, PnPs19A, PnPs19F, and PnPs23F. Each 0.5 mL dose contains approximately 2.2 pg of twelve serotypes and approximately 4.4 pg of serotype PnPs68. in addition, each dose contains approximately 32 pg CRM 97> 125 pg aluminum phosphate (adjuvant), 0.295 mg (buffer) and 0.02% polysorbate 80 (stabilizer). [0097] PREVNAR 13© is indicated for the active immunization for the prevention of invasive disease caused by the thirteen serotypes in children 6 weeks through 17 years of age (prior to the 18Th birthday) and in adults 50 years of age and older. PREVNAR 13© is also indicated for the aciive immunization for the prevention of otitis media caused by S. pneumonia serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.
[0098] ΊΎΡΗΙΜ VI© Typhoid Vi Polysaccharide Vaccine {Sanofi Pasteur SA) is a commercially available vaccine. The vaccine contains the cell surface Vi polysaccharide extracted from Salmonella enterica serovar Typhi, S. typhi Ty2 strain. Each 0.5 mL dose is formulated to contain 25 pg purified Vi polysaccharide in a colorless isotonic phosphate buffered saline (pH 7 ± 0.3), 4.150 mg sodium chloride, 0.065 mg disodium phosphate, 0.023 mg monosodium phosphate, and 0.5 ml sterile water for injection.
[0099] TYPHIM Vi® is indicated for the active immunization against typhoid fever for persons two years of age or older.
Example 13
Polysaccharide Content Detection ELISA Protocol PnPs Containing Samples
[00100] Virtual conjugate particles were analyzed for polysaccharide content.
[00101] Anti-PnP antibodies were diluted to 0.15pg/mL in 0.05M carbonate/bi-carbonate buffer (pH 9.6) and added at 100pL/weil to Maxisorb 98-vve!l plates, which were then sealed and incubated at 4 C overnight. The next morning, the coating solution was decanted and the plates were washed one time using washing buffer (1x PBS, 0.2% Tween-20). Blocking buffer (1x PBS, 2% BSA, 0.2% Tween-20) was added at 200pL/weli. The plates were sealed and incubated at 37°C for greater than or equal to two hours. [0100] Samples and standards were prepared during the blocking step. Particle samples and PnP standard (approximately 3-4mg/mL) were treated with an equal volume of Proteinase K enzyme (PK, stock 1 mg) for 40 minutes at 65°C with intermittent vortexing. Upon completion of the 40 minute incubation, a 1 :25 dilution of 0.1 M PefaBloc SC Plus was added to all samples and standards. Samples were treated in the order that they would be added to plates in the next step. After the PK treatments, the clarity of samples prior to diluting (cloudy, hazy, clear) was recorded. The treated standard was diluted in blocking buffer to the target starting concentration of 25 ng/mL then serially diluted 6 x 1/2 in blocking buffer. Treated samples were serially diluted 4 x 1/10 in blocking buffer.
[0101] Upon completion of sample treatment and a minimum blocking period of two hours, the blocking buffer was decanted and the plates washed one time with wash buffer. Samples and standards were added at 100 μΙ_Λ/νβΙΙ to the plates in duplicate. The plates were sealed and incubated at 37°C for 1 hour. The secondary antibodies (biotinylated-anti-PnP) were diluted to 0.2 μg/mL in blocking buffer. The plates were washed three times with wash buffer. The secondary antibody solution was added at 100 μΙ_Λ/νβΙΙ, and the plates were sealed and incubated at 37°C for 1 hour. Alkaline-phosphatase-conjugated streptavidin (SA-AP) was diluted 1 :1000 in blocking buffer. The plates were washed four times with wash buffer. SA-AP was added at 100 μΙ_Λ/νβΙΙ to the plates with either 40 or 60 seconds between each plate to ensure consistency in development. The plates were sealed and incubated at 37°C for 30 minutes.
[0102] PNPP substrate tablets were dissolved in ultrapure water (1 gold and 1 silver tablet per 20 ml_s of water) and passed through a 0.2 μηη filter. The plates were washed six times with wash buffer. PNPP substrate solution was added at 100 μΙ_Λ/νβΙΙ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the highest concentration of the standard curve to have an OD of approximately 1.0-1.5. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm (with purple plate adapter in place). Custom SoftMax Pro templates were used for data analysis.
[0103] Typhoid Polysaccharide Containing Samples
[0104] Anti-Vi antibodies were diluted 1 : 1000 in 0.0 5M carbonate/bi-carbonate buffer (pH 9.6) and added at 100 μΙ_Λ/νβΙΙ to Maxisorb 96-well plates, which were then sealed and incubated at 4°C overnight. The next morning, the coating solution was decanted and the plates washed one time with washing buffer (1x PBS, 0.2% Tween-20). Blocking buffer (1x PBS, 2% BSA, 0.2% Tween-20) was added at 200 μΙ_Λ/νβΙΙ. The plates were sealed and incubated at 37°C for greater than or equal to two hours.
[0105] Samples and standards were prepared during the blocking step. Preparation of two standards is required. First, a Vi-CRM GA crosslinked standard was read against all particle groups crosslinked with GA. Secondly, a Vi-CRM EDC crosslinked standard was read against all particle groups crosslinked with EDC. The standards were diluted in blocking buffer to the desired starting concentration of 1000 ng/mL then serially diluted 7 x 1/2 in blocking buffer. Samples were diluted 1/10 in blocking buffer for the first dilution. Then samples were serially diluted 3 x 1 /4 in blocking buffer.
[0106] Upon completion of sample dilutions and a minimum blocking period of two hours, the blocking buffer was decanted and the plates washed once with wash buffer. Samples and standards were added at 100 μΙ_Λ/νβΙΙ to the plates in duplicate. The plates were sealed and incubated at 37°C for 1 hour. The primary antibodies (rabbit polyclonal anti-Vi antibodies) were diluted 1 :200 in blocking buffer. The plates were washed three times using wash buffer. The secondary antibody solution was added at 100 μΙ_Λ/νβΙΙ, and the plates were sealed and incubated at 37°C for one hour. Alkaline-phosphatase-conjugated Donkey anti-Rabbit IgG (AP-
IgG) was diluted 1 :1000 in blocking buffer. The plates were washed four times using wash buffer. AP-lgG was added at 100 μΙ_Λ/νβΙΙ to the plates with either 40 or 60 seconds between each plate to ensure consistency in development. The plates were sealed and incubated at 37°C for one hour.
[0107] PNPP substrate tablets were dissolved in ultrapure water and passed through a 0.2μηη filter. The plates were washed six times using wash buffer. PNPP substrate solution was added at 100 μΙ_Λ/νβΙΙ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the highest concentration of the standard curve to have and OD of approximately 2.0. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm (with purple plate adapter in place). Custom SoftMax Pro templates were used for data analysis.
Example 14
Protein Analysis
[0108] Virtual conjugate particles were analyzed for protein content using the Protein Content Assay Bicinchoninic Acid (BCA) Protocol. Formulated particle suspensions were centrifuged to pellet the particles, with the resulting supernatant discarded. The pellets were reconstituted in phosphate buffered saline (PBS) and diluted to a working concentration. BCA Reagent was then added and the samples incubated at 37°C for a minimum of three hours. The resulting samples were then cooled to room temperature and read on a Spectramax M5 microplate reader at 562 nm. Data was analyzed by the instrument software.
Example 15
Immunological Testing: IgG ELISA Protocol [0109] Virtual conjugate particles were analyzed for immunological activity utilizing an ELISA assay.
[01 10] PnPs and PLD Containing Samples
[01 11] PnPs was diluted to 2 or 20 μg/mL or PLD was diluted to 2 μg/mL in 1X PBS (pH 9.6) and added at 50 \Uwe\\ to Maxisorb 96-well plates, which were then sealed and incubated at 4°C overnight. The next morning, the coating solution was decanted and the plates washed one time with washing buffer (1X PBS, 0.2% Tween-20). Blocking buffer (1X PBS, 2% BSA, 0.2% Tween-20) was added at 200 \UweW. The plates were sealed and incubated at 37°C for greater than or equal to two hours.
[01 12] PnPs Samples: Samples and controls were prepared during the blocking step. Serum samples were thawed at 4°C overnight. Fresh cell-wall polysaccharide/22F (CWPS/22F) absorption buffer (5 μg/mL CWPS, 5 μg/mL 22F, 0.05% Tween-20, 1X PBS) was prepared. Serum samples were diluted 1/40 in CWPS/22F absorption buffer, and control samples were diluted to a predetermined concentration in CWPS/22F absorption buffer. Samples were added to the dilution plate in the order than they would be added to plates in the next step. Samples and standard were incubated for one hour at room temperature. Samples were then diluted 10X 1/2 in CWPS/22F absorption buffer.
[01 13] PLD Samples: Samples and controls were prepared during the blocking step. Serum samples were thawed at 4°C overnight. Absorption buffer (0.05% Tween-20, 1X PBS) was prepared. Serum samples were diluted 1/40 in absorption buffer, and control samples were diluted to a predetermined concentration in absorption buffer. Samples were added to the dilution plate in the order than they would be added to plates in the next step. Samples were then diluted 10X 1/2 in absorption buffer. [01 14] The blocking buffer was decanted and the plates washed three times with wash buffer. Samples and controls were added at 50 μΙ_Λ/νβΙΙ to the plates in duplicate. The plates were sealed and incubated at 37°C for one hour. The secondary antibody was diluted in blocking buffer. For rabbits, the secondary antibody (alkaline-phosphatase-conjugated goat-anti-RABBIT IgG, subclasses 1 +2a+2b+3, FCy fragment specific) was diluted 1 :1000 in blocking buffer. For mice, the secondary antibody (alkaline-phosphatase-conjugated goat-anti-MOUSE IgG, subclasses 1 +2a+2b+3, FCy fragment specific) was diluted 1 :750 in blocking buffer. The plates were washed three times with wash buffer. Mouse PnPs IgG plates were washed four times. The secondary antibody solution was added at 50 μΙ_Λ/νβΙΙ to the plates with either 40 or 60 seconds between each plate to ensure consistency in development. The plates were sealed and incubated at room temperature for one hour.
[01 15] PNPP substrate tablets were dissolved in ultrapure water and passed through at 0.2 μιτι filter. The plates were washed three times with wash buffer. Mouse PnPs IgG plates were washed four times. PNPP substrate solution was added at 100 μΙ_Λ/νβΙΙ with either 40 or 60 seconds between each plate to ensure consistency in development. Plates were incubated at room temperature to allow the control samples to reach a predetermined optical density. The plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm. Custom SoftMax Pro templates were used for data analysis.
[01 16] Typhoid Polysaccharide
[01 17] The analysis of Typhoid Polysaccharide was derived from Nahm M and Goldblatt D. 26 November 2002 posting date, Training manual for enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (PnPg ELISA) and Wernette CM, et. al. 2003. Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides, Clin. Diagn. Lab. Immunol. 10:514-591.
[01 18] Solutions were prepared for use in sample analysis. [01 19] Coating Buffer 10X Stock (10X PBS/0.2% NaN3): 80 g NaCI, 3.14 g KH2P04, 20.61 g Na2HP04 ■ 7 H20, 1.6 g KCI, and 2 g NaN3 were dissolved in 1000 mL dH20. After dissolution, the solution was sterile filtered and stored at room temperature.
[0120] Coating Buffer (1X PBS/0/02%NaN3): 900 mL dH20 was added to 100 mL Coating Buffer 10X Stock. The pH was determined using an aliquot for the resulting solution. If the pH was not 7.2 +/- 0.2, the Coating Buffer 10X stock solution was discarded and a new solution was prepared.
[0121] Antibody Buffer 10X Stock (10X PBS/0.2% NaN3/0/5% Tween 20): 80 g NaCI, 3.14 g KH2P04, 20.61 g Na2HP04 ■ 7 H20, 1.6 g KCI, 2 g NaN3 were dissolved in 1000 mL dH20. 5 mL Tween 20 was added. After dissolution, the solution was sterile filtered and stored at room temperature.
[0122] Antibody Buffer (1X PBS/0.02% NaN3/0/05% Tween 20): 900 mL dH20 was added to 100 mL Antibody Buffer 10X Stock. The pH was determined using an aliquot from the resulting solution. If the pH was not 7.2 +/- 0.2, the Antibody Buffer 10X stock solution was discarded and a new solution was prepared.
[0123] Wash Buffer 10X Stock: 80 g NaCI, 1.6 g KCI, 0.94 g Trizma Base, 14.56 g Trizma HCI were weighed. dH20 was added to 800 mL. After dissolution, 33 mL Brij-35 (30% wt/v) was added and the solution was mixed thoroughly. The final volume was brought to 1000 mL using dH20. The solution was stored at room temperature for up to 12 months.
[0124] Wash Buffer: One part Wash Buffer 10X Stock was combined with nine parts dH20. The pH was verified to be 7.2 +/- using an aliquot of the resulting solution.
[0125] Stop Solution (3M NaOH): 120 g NaOH pellets were slowly added to 800 mL dH20 to control the generation of the resulting exotherm. After the dissolution was complete and the solution cooled to room temperature, the total volume was brought to 1000 ml. using dH20. the solution was stored at room temperature for up to 12 months.
[0126] Vi (Typhoid Polysaccharide) was diluted to 1 .25 μg/mL in Coating Buffer and added at 100 iUweW to medium binding, 96-well flat bottom, polystyrene plates (for example Greiner 655001 , or equivalent), which were then covered and incubated at 37°C for 5 hours. Plates were then cooled and stored at 4°C overnight until day of the assay (for up to 1 week). On the day of the assay, plates were brought to room temperature until samples were ready to be added.
[0127] Serum samples were thawed at 4°C overnight the night before the assay. Fresh Antibody Buffer was prepared from Antibody Bufferl Ox Stock. Serum samples were diluted 1/40 in Antibody Buffer, and control samples (positive and negative) were diluted to a predetermined concentration in antibody buffer. Samples were added in the order that they would be added to plates in the next step. Samples were then serial diluted two fold ten times in Antibody Buffer across a row.
[0128] Coating Buffer was then decanted and plates were washed with Wash Buffer. The first wash incubated for 1 minute, then plates were washed four more times for a total of five washes. Plates were then blotted dry on paper towel. Samples and controls were added at 50iUweW to the plates in duplicate. Plates were sealed and incubated at room temperature for approximately two hours.
[0129] The secondary antibody (alkaline-phosphatase-conjugated goat-anti-mouse IgG, γ-chain specific; for example Sigma-Aldrich No. A3438, or equivalent) was diluted 1 :8000 in Antibody Buffer. Plates were washed as described above for a total of five washes using Wash Buffer. The secondary antibody solution was added at 100 μΙ_Λ/νβΙΙ to the plates with two minutes between each plate to ensure consistency in development. The plates were covered and incubated at room temperature for two hours. [0130] Approximately 30 minutes prior to development, PNPP substrate tablets were dissolved in ultrapure water and passed through a 0.2 μηη filter. The plates were then washed as described above for a total of five washes using Wash Buffer. PNPP substrate solution was added at 100 μΙ_Λ/νβΙΙ with two minutes between each plate to ensure consistency in development. Plates were incubated at room temperature for two hours.
[0131] At the conclusion of two hours 50 μΙ_Λ/νβΙΙ 3M NaOH Stop Solution was added to each plate allowing two minutes between plates. After addition, each plate was tapped and allowed to sit for approximately seven minutes prior to reading. Plates were read on a SpectraMax M5 plate reader in absorption mode at 405 nm and again at 690 nm. Data reported was the difference between the readings at 405 nm and 690 nm. Custom Excel templates were used for data analysis.
Example 16
Immunological Testing: Opsonophagocytic Killing Assay (OPK)
[0132] Virtual conjugate particles were analyzed for immunological activity utilizing an OPK assay. Samples were testing according to the protocol detailed in Burton RL and Nahm MH. Development and validation of a fourfold multiplexed opsonization assay (UAB-MOPA) for pneumococcal antibodies. Clin. Vacc. Immuno. 2006; Sept; 13(9):1001-1009.
Example 17
Anti-RSV IgG ELISA
[0133] 96 well microtiter plates were coated with a protein solution containing either RSV F or HA protein at a concentration of approximately 2 μg/mL in carbonate-bicarbonate buffer. Plates were covered and incubated at 4°C overnight. The following morning the coating solution was removed from the plates and the plates were washed in 1X PBS using a plate washer. 200 μΙ_ Blocking Buffer (30 g BSA + 1 L 1X PBS) was added to all wells and the plates were covered and incubated for approximately 1-2 hours at 37°C.
[0134] Serum samples and control were prepared during the blocking incubation period. Serum samples were diluted in Reagent Diluent (10 g BSA + 500 μΙ_ Tween 20 + 1 L 1X PBS) to the desired starting concentration in a minimum total volume of 300 μΙ_. Samples were serially diluted 40 fold from column 1 to column 2, then serially diluted 2 fold from column 2 through column 1 1. A positive control was prepared by diluting anti-RSVF (monoclonal Ab, stock at approximately 1 mg/mL) 1 : 1000 by adding 10 μί Ab into 10 mL of Reagent Diluent (final concentration approximately 31.3 ng/mL). Immediately prior to adding samples, the Blocking Buffer was removed and the plates were washed three times with 1X PBS using a plate washer. 50 [iL of diluted serum sample per well was transferred to the ELISA plate. Samples were run in duplicate. The plate was covered and incubated at 37°C for approximately one hour.
[0135] Secondary antibody was prepared at a dilution of 1 :1000 by adding 200 μί Ab into 200 mL of Assay Diluent (AP-conjugated Goat Anti-mouse IgG (subclasses 1 + 2a + 2b + 3), Fc-γ fragment specific (Jackson I mmu no Research catalog 115-055-164)). Plates were washed three times with 1X PBS using a plate washer. The plates were blotted dry. 50 \Uwe\\ of secondary antibody was added to each plate at 40 second intervals and the plates were incubated at room temperature for approximately one hour.
[0136] Developer was prepared using SIGMAFAST tablets (p-nitrophenyl phosphate) (Sigma catalog N2770-50SET). 1 TRIS buffer tablet and 1 pNPP tablet were added per 20 mL water. Plates were washed three times using 1X PBS in a plate washer. Plates were blotted dry and Developer was added at 100 \Uwe\\ to each plate at 40 second intervals. Plates were incubated at room temperature to allow the control samples to reach a predetermined optical density. The plates were read usinga Spectromas M5 plate reader at an absorbance of 405 nm every five minutes beginning at T = 25-30 minutes. Custom SoftMax Pro templates were used for data analysis.
Example 18
ELISpot Procedure
[0137] Prepare Red Blood Cell Lysis Solution by dissolving approximately 8.29 g ammonium chloride, 1.0 g potassium bicarbonate, and 0.0372 disodium EDTA dehydrate in 900 mL deionized water. Adjust the pH to 7.2 to 7.4 using sodium hydroxide. Add deionized water to a final volume of 1 L and sterile filter. Store at room temperature.
[0138] 70% ethanol was prepared from 100% ethanol using sterile water. Coating solutions were prepared by diluting mouse IL-2 (interleukin-2) 1 :200 with sterile 1X PBS (0.275 mL into 54.725 mL) and by also diluting mouse IFN-gamma (interferon-gamma) 1 :200 with sterile 1X PBS (0.275 mL into 54.725 mL). Membranes of the MultiScreen-IP Hydrophobic PVDF 96 well plates were pre-wetted by adding 15 \Uwe\\ of 70% ethanol for 50-60 seconds. The ethanol solution was decanted and the plates were washed three times with 1X PBS using 150 \UweW. Next the plates were coated by adding 100 μί of the appropriate coating solution and incubate overnight at 4°C.
[0139] The following day, the coating solution was decanted and the plates were washed three times with cRPMI medium (10% fetal bovine serum/1 % Pen/strep/1 % nonessential amino acids/1 % sodium pyruvate/25 mM Hepes/0.1 % BME) leaving the last wash as a blocking medium. The plates were then incubated at room temperature for at least two hours.
[0140] The splenocytes were prepared. Spleens were mashed gently and spun at 450 X g at room temperature for approximately 5 minutes. The medium was removed and the tube scraped to loosen the cells. Next, 10 mL of RBC lysis solution was added to the tube and the tube was inverted several times. The tube was spun again at 450 x g at room temperature for approximately 5 minutes. The supernatant was removed and the tube scraped to loosen the cells. Next, 15 mL of cRPMI medium was added. The suspension was poured through a 40 μηη cell strainer into a sterile 50 mL conical tube. The filtered suspension was transferred into a fresh 15 mL conical tube and centrifuged a final time at 450 x g at room temperature for approximately 5 minutes. The medium was decanted leaving approximately 0.5 to 1.0 mL medium with the cells.
[0141] The trypan blue cell stain was prepared by adding 2 μΙ of the splenocytes to 498 μί of 0.1 % trypan blue. The cells were counted in each of the four corner quadrants of the hemacytometer. Also, based on cell count 3e6 cells/mL to a total volume of 3.5 mL for each sample were prepared.
[0142] Various stimulation media were prepared. Working solutions of PMA, ionomycin, and nucleic acid peptide were prepared. For PMA, a stock solution of approximately 2 mg/mL was diluted 1 :40 in cRPMI medium by adding 10 μί of stock PMA into 390 μί of cRPMI. For ionomycin, a stock solution of approximately 10 mM and 7.14 mg/mL was diluted 1 :10 in cRPMI medium by adding 15 μί of stock into 135 μί of cRPMI. For HA-RNA peptides, vials were prepared at approximately 100 μg/mL. For RSV-F RNA peptide, vials were prepared at approximately 100 μg/mL.
[0143] All simulations were prepared at 10X the final concentration to account for the dilution into cells. Positive stimulation medium was prepared by combining approximately 30 μί working solution of PMA, 30 μί working solution of ionomycin, 30 μί of alpha CD28, and 2.1 mL of cRPMI medium.
[0144] Negative stimulation medium was prepared by combining approximately 50 μί of alpha CD28 and 4.95 mL of cRPMI medium. [0145] HA RNA peptide stimulation medium was prepared by combining approximately 0.6 mL of working HA-RNA peptide solution, 30 μΙ_ alpha CD28, and 2.37 mL cRPMI medium.
[0146] RSV-F RNA peptide stimulation medium was prepared by combining approximately 0.2 mL of working RSV-F RNA peptide solution, 10 μί alpha CD28, and 0.79 mL cRPMI medium.
[0147] Cell dilution plates were prepared according to the planned layout. Appropriate controls were incorporated. Stimulated cells were added to the blocked plates at 100 \Uwe\\. Plates were incubated at 37°C and 5% C02 overnight undisturbed.
[0148] Buffers were prepared. Wash buffer I was prepared by adding 0.05% Tween 20 to 1X PBS. Wash buffer II was 1X PBS. The assay diluent was 1X PBS with 10% FBS.
[0149] Following the overnight incubation period, the cell suspension was decanted from the plates. The plates were washed two times using deionized water with a 3-5 minute soak for each water wash. Next, the plates were washed three times with wash buffer I. The detection antibody (Anti IL-2 and Anti IFN gamma) was diluted 1 :250 in assay diluent (10% fetal bovine serum in 1X PBS)by adding 0.22 mL of antibody into 54.88 mL of buffer for each cytokine. The detection antibody was added at 100 \Uwe\\ and the plates were covered and allowed to incubate at room temperature in the dark for approximately two hours.
[0150] The detection antibody solution was discarded and the plates were washed three times with wash buffer I. Next, streptavidin-horseradish peroxidase was diluted 1 :100 in assay diluent by combining approximately 1.1 mL HRP with 108.9 mL of assay diluent. Plates were washed three times with wash buffer I. HRP was added at 100 \Uwe\\ and the plates were covered and allowed to incubate at room temperature in the dark for approximately one hour.
[0151] Approximately fifteen minutes prior to the end of the HRP incubation period substrate solution was prepared by adding 1 drop (approximately 30 μί) of AEC Chromogen per 1 mL of AEC substrate. The substrate solution was vortexed thoroughly and stored in the dark at room temperature until use.
[0152] Plates were removed from the dark and the HRP solution discarded. The plates were washed four times with wash buffer 1 and two times with wash buffer II. To develop plates, 100 iUweW of substrate solution was added. The plates were allowed to develop until the desired color was achieved, generally 30-60 minutes. Once the desired color was achieved the plates were submerged in deionized water. The plastic backing was then removed and the plates were re-submerged in fresh deionized water. Excess water was removed and the plates were dried overnight in the dark.
[0153] Once dry, membranes were punched out onto adhesive. The location of A1 was identified for the plate. Samples were sent to an external vendor for evaluation.
Example 19
Pneumococcal Polysaccharide Mouse Study #1
[0154] A series of virtual conjugate particles were produced substantially according to Example 5 for injection into mice. For this study, approximately 100 μg of the pharmaceutal composition in approximately 500 μΙ_ vehicle was injected. Injections were placed in the dorsal thoracic region using a 21 gauge needle. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
Figure imgf000054_0001
14
51- 5.0 5.0 2.0 1.35 CRM 4.0 1 1.64 GA 1.75 15
51- 5.0 5.0 2.0 1.35 PLD 4.0 9.71 GA 1.75 16
51- PREVNAR 13® None
26
[0155] Samples were produced using the core particle of Example 1 (PLGA, 80 nm X 320 nm). All samples for this study were cross-linked utilizing glutaraldehyde.
[0156] A mass of particles fabricated by any of Example 1 were aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL A 5% (5 mg/mL in water) protamine sulfate solution was added and the suspension was rocked at room temperature overnight. To wash away unbound protamine, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 μΙ_ water until no particle chunks were present. The volume was increased to 1 ml. to continue to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re- suspended to a targeted concentration, for example 2 mg/mL, in water.
[0157] Polysaccharide and protein were added to the particle suspension using aqueous stock solutions. The polypropylene tube was placed on a rocker for one hour at room temperature.
After the one hour incubation period, glutaraldehyde was added. The cross-linking time was approximately twelve hours at room temperature with rocking. 35mM sodium cyanoborohydride
(NaCNBH3) was added to quench the reaction. The tube was rocked one hour at room temperature. To wash away unbound components, the suspension was centrifuged at 12,500 X g for 30 minutes and the supernatant was discarded. The pellet was sonicated in 300 to 500 μΙ_ water until no particle chunks were present. The volume was increased to 1 ml. water to continue to wash the particles. The suspension was centrifuged a second time at 12,500 X g for 30 minutes. The wash step was repeated a second time and the suspension was centrifuged a third time at 12,500 X g for 30 minutes. The supernatant was discarded and the pellet was re- suspended to a targeted concentration, for example 2 mg/mL, in 0.1 % polyvinyl alcohol/5% mannitol.
[0158] The polysaccharide content was determined according to Example 13. The protein content was determined according to Example 14. The table below contains the test results.
Figure imgf000056_0001
[0159] Immunological testing was conducted according to Example 15 and results are shown in Figure 8. Figure 8 shows the Anti-PnPs14 IgG Reciprocal Titer for each sample at four points in time: pre-bleed, post-prime, post-boost 1 , and post-boost 2. As a note, Sample 51-15 was primed one week after the other two groups, so post-prime data was collected at three weeks instead of four weeks like the other samples.
[0160] The data shows that each pharmaceutical composition produced had immunological activity greater than or equal to that of PREVNAR 13®. The pharmaceutical composition showed immunological activity approximately equivalent to PREVNAR 13® when PnPs14 was combined with OVA, CRM197, or PLD when using glutaraldehyde as the cross-linker.
Example 20
Pneumococcal Polysaccharide Mouse Study #2
[0161] A series of virtual conjugate particles were produced for implantation into mice. For this study, approximately 100 μg of the pharmaceutal composition in approximately 500 μΙ_ vehicle was injected. Injections were placed in the dorsal thoracic region using a 21 gauge needle. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
Figure imgf000057_0001
55- 0.0 0.0 PnPsl 2.0 5.33 PLD 4.0 3.50 BMH 2X mass 14 carrier protein
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 None 0 15
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 None 0 16
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 GA 0.7 17
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 GA 0.7 18
55- 5.0 5.0 PnPsl 2.0 5.15 PLD 4.0 3.50 GA 0.7
19 Sized
55- 5.0 5.0 PnPsl 2.0 2.24 PLD 4.0 3.50 GA 0.7 20 ATCC
55- 5.0 5.0 PnPsl 2.0 5.33 CRM 4.0 3.50 GA 0.7 21
55- 5.0 5.0 PnPs5 2.0 4.98 PLD 4.0 3.50 GA 0.7 22
55- 5.0 5.0 PnPs5 2.0 4.04 PLD 4.0 3.50 GA 0.7 23 Sized
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 EDC 10X 24 mass carrier protein
55- 5.0 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 BMH 2X mass 25 carrier protein
55- 2.5/2.5 5.0 PnPsl 2.0 5.33 PLD 4.0 3.50 GA 0.7 26
[0162] Samples 55-6 through 55-1 1 , 55-13, and 55-14 utilized the core particle of Example 3 (PLGA/DC-Chol). Samples 55-15 through 55-26 utilized the core particle of Example 1 (PLGA, 80 nm X 320 nm). This animal study examined various polysaccharides, cross-linkers, and processing methods. [0163] Samples 55-7 through 55-1 1 were produced according to Example 7. Samples 55-18 through 55-23 were produced according to Example 5.
[0164] Sample 55-13 and 55-24 were produced substantially according to Example 8 and Example 6 respectively. When removing the unbound protamine and protein, the particles were washed once instead of twice.
[0165] Samples 55-14 and 55-25 were produced substantially according to Example 7 and Example 5 respectively. Instead of adding glutaraldehyde as described in Examples 7 and 5, bismaleimidohexane (in DMSO) was added to the suspension. After agitation for one hour, the suspension was filtered with a 1 1 μιτι filter to remove any crystalized BMH. The virtual conjugate particles were then washed twice as described.
[0166] Samples 55-6 and 55-15 were produced as follows. A mass of particles fabricated by Example 1 (55-15) or Example 3 (55-6) was aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL Protamine was added to 55-15. Polysaccharide and protein were added to the particle suspension using stock solutions. The suspension was agitated for one hour at room temperature. No cross- linker was used. The resulting virtual conjugate particles were washed twice as described above.
[0167] Samples 55-16 and 55-17 were produced substantially according to Example 5. However, prior to washing the particles after the polysaccharide and protein additions, additional protamine was added to the suspension. The suspension was agitated for one hour at room temperature and the resulting virtual conjugate particles washed twice as described above. Sample 55-17 was then cross-linked using glutaraldehyde as follows. Glutaraldehyde solution was added to the re-suspended particles and agitated for one hour at room temperature. Sodium cyanobrohydride was added to quench the reaction and the virtual conjugate particles were washed twice as described above. [0168] Sample 55-26 was produced as follows. A mass of particles fabricated by Example 1 were aliquoted into protein Lo-bind polypropylene tubes and water was added to achieve a targeted concentration, for example 2 mg/mL Protamine solution was added and the suspension was incubated at room temperature for ten minutes. Polysaccharide was added and the suspension was incubated at room temperature for ten minutes. Protein was added and the suspension was incubated at room temperature for ten minutes. The remaining protamine was added and followed immediately with the addition of glutaraldehyde. After agitation for one hour at room temperature, the glutaraldehyde was neutralized with sodium borohydride and the resulting virtual conjugate particles washed twice as described above.
[0169] The polysaccharide content was determined according to Example 13. The protein content was determined according to Example 14. The table below contains the test results.
Figure imgf000060_0001
-1 1-P2B 0.14 4.00 0.04-13-PP 1.42 3.00 0.47-13-P1 B 1.32 3.00 0.44 0.44-13- P2B 1.23 3.00 0.41-14-PP 1.75 3.00 0.58-14-P1 B 1.01 3.00 0.34 0.42-14-P2B 1.02 3.00 0.34-15-PP 0.77 3.00 0.26-15-P1 B 0.48 3.00 0.16 0.28-15- P2B 1.73 4.00 0.43-16-PP 2.77 5.00 0.55-16-P1 B 2.49 4.00 0.62 0.51-16- P2B 1.79 5.00 0.36-17-PP 0.1 1 5.00 0.02-17-P1 B 0.06 5.00 0.01 0.02-17- P2B 0.07 3.00 0.02-18-PP 0.57 3.00 0.19-18-P1 B 0.57 3.00 0.19 0.17-18- P2B 0.42 3.00 0.14-19-PP 0.37 3.00 0.12-19-P1 B 0.28 3.00 0.09 0.11-19- P2B 0.40 4.00 0.10-20-PP 0.59 3.00 0.20-20-P1 B 0.41 3.00 0.14 0.15-20-P2B 0.34 3.00 0.1 1-21-PP 0.04 3.00 0.01-21-P1 B 0.02 2.00 0.01 0.01-21-P2B 0.04 2.00 0.02-22-PP 0.84 4.00 0.21-22-P1 B 0.48 3.00 0.16 0.18-22-P2B 0.69 4.00 0.17-23-PP 0.21 4.00 0.05-23-P1 B 0.1 1 3.00 0.04 0.05-23-P2B 0.24 4.00 0.06-24- PP 1.04 4.00 0.26-24-P1 B 1.44 3.00 0.48 0.34-24- P2B 1.15 4.00 0.29-25-PP 0.70 3.00 0.23-25-P1 B 0.78 3.00 0.26 0.33-25-P2B 1.96 4.00 0.49-26-PP 0.01 4.00 0.00 0.00 55-26-P1 B 0.02 4.00 0.01
55-26-P2B 0.02 4.00 0.01
[0170] Immunological activity was evaluated according to Example 15.
[0171] Figure 9 shows Anti-PnPs1 IgG Reciprocal Titer for samples utilizing the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm). When comparing PnPsl with the PLGA DC- Chol core particle and various cross-linker types, glutaraldehyde (55-7) produced a higher titer than EDC (55-13) which produced a higher titer than BMH (55-14). When comparing the two carrier proteins with glutaraldehyde as a cross-linker, PLD (55-7) and CRM197 (55-9) were approximately equivalent. When comparing sized polysaccharide to native polysaccharide, sized polysaccharide (55-8) provided a higher titer than native polysaccharide (55-7) for PnPsl , PLD carrier protein, and glutaraldehyde cross-linker. Also of note, the non-cross-linked sample (55-6) produced a lower titer than the control. In summary, when using the PLGA DC-Choi core particle with PnPsl , glutaraldehyde was an acceptable cross-linker with either PLD or CRM197 as a carrier protein. Using sized polysaccharide further boosted the titer response.
[0172] Figure 10 shows Anti-PnPs1 IgG Reciprocal Titer for samples utilizing the particle of
Example 1 (PLGA, 80 nm X 320 nm). When comparing PnPsl with the protamine core particle and various cross-linkers, EDC (55-24) produced a higher average titer than glutaraldehyde (55-
18) which produced a higher average titer than BMH (55-25). When comparing the two carrier proteins with glutaraldehyde as a cross-linker, PLD (55-18) produced a higher titer response than CRM197 (55-21 ). When comparing sized polysaccharide to native polysaccharide, sized polysaccharide (55-19) was approximately equal to native polysaccharide (55-18) for PnPsl and glutaraldehyde cross-linker. When comparing the two polysaccharide vendor sources, SI I had a slight edge and both were approximately equivalent to PREVNAR 13®. Also of note, the non-cross-linked sample (55-15), the double coated sample (55-16), and the "one pot" sample
(55-26) produced a lower titer than the PREVNAR 13® control. Performance of the double coated sample (55-16) was improved with cross-linking (55-17). Samples with performance equal to the PREVNAR 13® control included: GA cross-linked samples (55-18, 55-19, 55-20, and 55-21 ) and the EDC cross-linked sample (55-24).
[0173] In summary, using the PLGA protamine core particle with PnPsl , there were several successful strategies. Selection of either EDC or glutaraldehyde as a cross-linker, either PLD or CRM197 as a carrier protein, or sized or native polysaccharide provided titer levels greater than or equal to that of control. When not using a cross-linker or when using a "one pot" strategy the pharmaceutical composition produced was less effective.
[0174] Figure 9 and Figure 10 establish that while the various combinations disclosed herein are effective, certain combinations of carrier proteins and cross-linkers provide particles that induce a greater titer. For example, for PnPsl , the PLGA protamine particle provided a greater titer response than the PLGA DC-Choi particle.
[0175] Figure 1 1 shows Anti-PnPs5 IgG Reciprocal Titer for samples 55-10, 55-1 1 , 55-22, and 55-23. In this example the use of native and sized PnPs5 polysaccharide was examined. In all cases the carrier protein was PLD and the cross-linker was glutaraldehyde. When using the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm), both native (55-10) and sized (55-11 ) PnPs5 performed approximately equal to PREVNAR 13® control. When using the particle of Example 1 (PLGA, 80 nm X 320 nm), both native (55-22) and sized (55-23) performed approximately equal to PREVNAR 13® control.
Example 21
Pneumococcal Polysaccharide Mouse Study #3
[0176] A series of virtual conjugate particles were produced for implantation into mice. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations. ID Protamine Polysaccharide Protein Cross-linker
% Stock, Serotype % Stock, Type % Stock, Type % mg/mL mg/mL mg/mL
56- PREVNAR 13® None 01
56- 0.0 0.0 NA NA NA PLD 4.0 4.37 None 0 03
56- 0.0 0.0 PnPsl 2.0 5.07 PLD 4.0 4.37 GA 0.35 10 Sized
0.0 0.0 PnPs5 2.0 3.99 PLD 4.0 4.37 GA 0.35
Sized
0.0 0.0 PnPs14 2.0 1.42 PLD 4.0 4.37 GA 0.35
56- 5.0 5.0 PnPsl 2.0 5.07 PLD 4.0 4.37 GA 0.7 18 Sized
5.0 5.0 PnPs5 2.0 3.99 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs14 2.0 1.42 PLD 4.0 4.37 GA 0.7
56- 5.0 5.0 PnPsl 2.0 5.07 PLD 4.0 4.37 EDC 10X 25 Sized mass carrier protein
5.0 5.0 PnPs5 2.0 3.99 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs14 2.0 1.42 PLD 4.0 4.37 GA 0.7
[0177] The purpose of this study was to evaluate the efficacy of a trivalent composition. For this animal study, three separate groups of particles each containing a different serotype were manufactured and then combined to produce a trivalent composition. To produce the trivalent composition, a volume containing approximately 100 μg of virtual conjugate particles from each of the various serotype compositions are combined. These samples were injected subcutaneously in the dosal thoracic region.
[0178] Sample 56-10 utilized the particle of Example 3 (PLGA DC-Chol, 80 nm X 320 nm) and followed Example 7. Sample 56-18 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5. Sample 56-25 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5 for PnPs5 (sized) and PnPs14 (native) and followed Example 6 for PnPsl (sized). After the separate virtual conjugate particles were produced, the various serotypes were formulated into a trivalent composition by combining the various serotypes. A volume containing approximately 100 μg of virtual conjugate particles from each of the various serotype compositions were combined.
[0179] A control sample (56-03) using protein only was prepared by dispersing PLD in 0.1 % polyvinyl alcohol/5% mannitol at a concentration of 0.012 mg/0.50 ml_.
[0180] Immunological activity was evaluated according to Example 15. The immunological activity was evaluated for each serotype independently (PnPsl , PnPs5, and PnPs14) as well as the immune response against PLD. All samples utilized PLD as the carrier protein, sized PnPsl polysaccharide, sized PnPs5 polysaccharide, and native PnPsl 4 polysaccharide.
[0181] Figure 12 shows Anti-PnPs1 IgG Reciprocal Titer for the three samples. Regarding PnPsl , the trivalent composition using the EDC cross-linker for PnPsl (56-25) produced the highest titer level. Next, the trivalent composition using glutaraldehyde and the PLGA protamine particle (56-18) produced titer equivalent to that of the PREVNAR 13® control. The trivalent composition using glutaraldehyde and the PLGA DC-Choi particle (56-10) produced an immunological response that was below that of the control. Figure 13 shows Anti-PnPs5 IgG Reciprocal Titer for the three samples. Regarding PnPs5, the trivalent composition using the EDC cross-linker for PnPsl (56-25) again produced the highest titer level. Next, the trivalent composition using glutaraldehyde and the PLGA protamine particle (56-18) produced titer greater than that of the PREVNAR 13® control. The trivalent composition using glutaraldehyde and the PLGA DC-Choi particle (56-10) produced an immunological response also greater than the control. Figure 14 shows Anti-PnPs14 IgG Reciprocal Titer for the three samples. Regarding PnPsl 4, the trivalent composition using the EDC cross-linker for PnPsl (56-25) again produced the highest titer level, just slightly above that of the PREVNAR 13® control. Next, the trivalent composition using glutaraldehyde and the PLGA protamine particle (56-18) produced titer slightly below than that of the control. The trivalent composition using glutaraldehyde and the PLGA DC-Chol particle (56-10) produced an immunological response that was below that of the control.
[0182] Figure 15 shows Anti-PLD IgG Reciprocal Titer for the three samples. The soluble PLD control produced the highest titer level. However, the trivalent composition using the EDC cross-linker for PnPsl (56-25) again produced the highest titer level, although below that of the soluble control. The two glutaraldehyde particles showed approximately equivalent immune response.
Example 22
Pneumococcal Polysaccharide Rabbit Study
[0183] A series of virtual conjugate particles were produced for implantation into New Zealand White rabbits. Samples were injected via two routes: subcutaneous (SC) and intramuscular (IM). The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
Figure imgf000066_0001
57-07 0.0 0.0 PnPsl 4.0 5.07 PLD 4.0 4.37 GA 0.35
Sized
0.0 0.0 PnPs5 4.0 3.99 PLD 4.0 4.37 GA 0.35
Sized
0.0 0.0 PnPs14 4.0 1.42 PLD 4.0 4.37 GA 0.35
57-08 5.0 5.0 PnPsl 4.0 5.07 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs5 4.0 3.99 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs14 4.0 1.42 PLD 4.0 4.37 GA 0.7
57-09 5.0 5.0 PnPsl 4.0 5.07 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs5 4.0 3.99 PLD 4.0 4.37 GA 0.7
Sized
5.0 5.0 PnPs14 4.0 1.42 PLD 4.0 4.37 GA 0.7
[0184] For this animal study, three separate groups of particles were made and then combined to produce a trivalent composition. Samples 57-06 and 57-07 utilized the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm) and followed Example 7. Samples 57-08 and 57-09 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5. Sample 57-05 utilized the particle of Example 1 (PLGA, 80 nm X 320 nm) and followed Example 5 for both PnPs5 (sized) and PnPsl 4 (native) and followed Example 6 for PnPsl (sized).
[0185] After the separate virtual conjugate particles were produced, the various serotypes were formulated into a trivalent composition by combining the various serotypes based on delivering a targeted dose of antigen. The polysaccharide content for the virtual conjugate particle samples were analyzed according to Example 13. Once the polysaccharide content was known, the composition was prepared to deliver a target dose of 2.2 μg per serotype.
[0186] Samples administered via subcutaneous injection included 57-01 , 57-05, 57-06, and 57- 08. Subcutaneous injections were delivered to the dorsal thoracic region. A total of 1.0 mL was delivered using a 21 gauge needle. Samples administered via intramuscular injection included 57-02, 57-07, and 57-09. Intramuscular injections were delivered in the quadriceps muscles located on the cranial aspect of the femur or the hamstrings on the caudal aspect of the femur using a 23 gauge needle. A total of 1.0 ml. was delivered.
[0187] Immunological activity was evaluated according to Example 15 and Example 16.
[0188] Figure 16A shows Anti-PnPs1 OPK Reciprocal Titer and Figure 16B shows Anti-PnPs1 IgG Reciprocal Titer Figure 17A shows Anti-PnPs5 OPK Reciprocal Titer and Figure 17B shows Anti-PnPs5 IgG Reciprocal titer. Figure 18A shows Anti-PnPs14 OPK Reciprocal Titer and Figure 18B shows Anti-PnPs14 IgG Reciprocal Titer.
[0189] Responses to all three serotypes were equivalent to the PREVNAR 13® control in at least one of the PLGA/protamine systems (see 57-05 in Fig. 16B) showing the virtual conjugate particles effective in a larger species (rabbit). Utilizing EDC as a cross-linker with PnPsl (57- 05) improved performance over the glutaraldehyde composition (57-08). When comparing routes of administration, performance, as measured by IgG response, appears to be dependent on both particle type and serotype. For PnPsl , SC (55-06) and IM (55-07) showed similar performance for PLGA DC-Choi based particles while IM (57-09) produced an immune response greater than or equal that of SC (57-08) for PLGA/protamine based particles. For PnPs5, IM (57-07) produced an immune response greater than or equal to that of SC (57-06) for PLGA DC-Choi based particles while IM (57-09) produced an immune response greater than that of SC (57-08) for PLGA/protamine based particles. For PnPs14, SC (57-06) produced an immune response greater than that of IM (57-07) for PLGA DC-Choi based particles while SC (57-08) produced an immune response greater than or equal to that of IM (57-09) for PLGA/protamine based particles.
Example 23
Typhoid Mouse Study [0190] A series of virtual conjugate particles were produced for implantation into mice. The table below lists the sample IDs, targeted component percentages, and aqueous stock solution concentrations.
Figure imgf000069_0001
[0191] Samples 59-04 and 59-05 utilized the core particle of Example 3 (PLGA/DC-Chol) and were fabricated according to Example 7. Samples 59-14, 59-15, 59-16, 59-21 , and 59-22 utilized the core particle of Example 1 (PLGA, 80 nm X 320 nm). Sample 59-14 was fabricated substantially according to Example 5 with the omission of glutaraldehyde. Samples 59-15 and 59-16 were fabricated according to Example 5. Samples 59-21 and 59-22 were fabricated according to Example 6. [0192] Sample 59-13 utilized the particle of Example 4 (PLGA DMAEMA). All samples were lyophilized with the exception of 59-04 and 59-15. Lyophilized samples were reconstituted in 0.1 % polyvinyl alcohol/5% mannitol prior to inoculation at a target concentration of 0.3 mg/0.5 ml_.
[0193] The polysaccharide content was determined according to Example 13. The protein content was determined according to Example 14. The table below contains the test results.
PS CP Ratio
ID ug/dose ug/dose PS/CP Ave
59-3-PP 2.00 0.00 NA
59-3-P1 B 1.70 0.00 NA NA
59-3-P2B 1.90 0.00 NA
59-4-PP 1.90 8.50 0.22
59-4-P1 B 2.20 13.30 0.17 0.20
59-4-P2B 3.20 15.50 0.21
59-6-PP 1.70 19.70 0.22
59-6-P1 B 2.10 23.70 0.17 0.20
59-6-P2B 2.10 23.70 0.21
59-12-PP 0.40 5.00 0.08
59-12-P1 B 0.50 4.00 0.13 0.12
59-12-P2B 0.40 2.80 0.14
59-13-PP 0.70 6.40 0.1 1
59-13-P1 B 0.30 6.00 0.05 0.07
59-13- P2B 0.20 5.40 0.04
59-14-PP 0.90 2.70 0.33
59-14-P1 B 0.50 3.00 0.17 0.36
59-14-P2B 1.30 2.30 0.57
59-15-PP 0.70 4.10 0.17
59-15-P1 B 0.10 4.90 0.02 0.20
59-15- P2B 1.60 3.90 0.41
59-16-PP 1.00 4.60 0.22
59-16-P1 B 1.30 6.10 0.21 0.32
59-16- P2B 1.70 3.30 0.52
59-21-PP 1.30 4.10 0.32
59-21-P1 B 4.90 4.50 1.09 0.64
59-21-P2B 1.50 2.90 0.52
59-22-PP 1.60 3.60 0.44
0.46
59-22-P1 B 1.50 4.10 0.37 59-22-P2B
[0194] Immunological activity was evaluated according to Example 15.
[0195] Figure 19 shows Anti-Vi IgG Reciprocal Titer for the tested samples that included protamine. Figure 20 shows Anti-Vi IgG Reciprocal Titer for the tested samples that included DC-Choi or DMAEMA.
[0196] The study showed lyophilized formulations are still functional for both the PLGA/protamine (59-15 v 59-16) system and the PLGA/DC-Chol system (59-04 v 59-05). The study also showed that EDC cross-linked samples are the highest responders for both core particle systems with the PLGA/protamine system providing the highest titer response.
[0197] The virtual conjugate particles proved to be a viable composition to deliver typhoid polysaccharide.
Example 24
RNA Mouse Study
[0198] A series of virtual conjugate particles were produced for implantation into mice. The table below lists the samples IDs and the source of the samples. Five animals were provided for each sample.
Figure imgf000071_0001
[0199] Two immunizations were given four weeks apart (prime and boost). A total of 120 μΙ_ was administered to each animal: 20 μΙ_ each rear footpad and 40 μΙ_ each rear thigh (IM). Immunological activity was assessed at T = 4 weeks (seven days post-boost) using ELISpot according to Example 18 and titer assessment using IgG ELISA according to Example 17.
[0200] Figure 21A-C shows the results of the immunological testing for the HA-RNA containing samples. Figure 21 A shows the T cell responses (IL2) in spots per million cells. Figure 21 B shows the T cell responses (IFNg) in spots per million cells. Figure 21 C shows the Anti-Flu HA IgG ELISA Titer for the tested samples.
[0201] Figure 22A-C shows the results of the immunological testing for the RSV-F RNA containing samples. Figure 22A shows the T cell responses (IL2) in spots per million cells. Figure 22B shows the T cell responses (IFNg) in spots per million cells. Figure 22C shows the Anti-RSV-F IgG ELISA Titer for both the prime and boost samples.
[0202] The data showed robust T cell responses (IFNg and IL2) for all samples tested. The data also showed humoral responses for all samples tested.
[0203] The virtual conjugate particles proved to be a viable composition to deliver nucleic acids such as RNA.
Example 25
Pneumococcal Polysaccharide Mouse Study #4, Study No. 64 as shown in Figure 23.
[0204] A series of virtual conjugate particles were produced for implantation into mice. The table below lists the sample IDs, target component percentages, and aqueous stock solution concentrations.
Group Protamine Polysaccharide Protein Cross-linker
% Stock, Serotype % Stock, Type % Stock, Type %
mg/mL mg/mL mg/mL
01 PREVNAR 13® None 02 Soluble PLD, PnPs 6A, PnPs 14A None
03 Soluble PLD, PnPs 6A, PnPs 14A, Alydrogel None
5.0 5.0 PnPs 6A 2.0 6.83 PLD 4.0 4.42 ADH 1 X mass of
PnPs
EDC 5X mass of carrier protein
33
5.0 5.0 PnPs 19A 2.0 8.21 PLD 4.0 4.42 ADH 1 X mass of
PnPs
EDC 5X mass of carrier protein
[0204] Samples 01 , 02, and 03 provided controls for the study: 01 : PREVNAR®, 02: soluble PLD/PnPs 6A/PnPs 14A, and 03: soluble PLD/PnPs6A/PnPs 14A and Alydrogel.
[0205] Sample 33 utilized the particle of Example 3 (PLGA DC-Choi, 80 nm X 320 nm). Particles were produced using either of following procedures.
[0206] Protamine and PLD were admixed and incubated at room temperature for 1 hour. The admixture was combined with the particles of Example 3 and rocked at room temperature for 1 hour. While the particles were rocking, PnPs 6A or PnPs 19A was combined with ADH and half the volume of EDC for 20 minutes. After 1 hour of rocking, the particles/protamine/PLD was pelleted to recover the protamine/PLD particles. The PnPs/ADH/EDC mixture was added to the pelleted protamine/PLD particles and the remaining volume of EDC was added. The mixture was combined using a pipet for 2 minutes. The mixture was brought to volume using buffer and the reaction was allowed to proceed for 2 hours at room temperature. After 2 hours, the resulting particles were pelleted and washed a total of three times using water (4 total spins).
[0207] The volume of PnPs 6A or PnPs 19A was doubled using 100 mM sodium acetate, pH
4.5. Adipic dihydrazide (ADH) and sodium cyanoborohydride (NaCNBH3) were added to the polysaccharide and the mixture was incubated at room temperature for 1 hour. PLD and the
PnPs mixture were added to pelleted protamine particles. EDC was added and the mixture was brought to volume with buffer. The reaction proceeded overnight at room temperature. The resulting particles were pelleted and washed a total of three times with water (4 spins).
[0208] Immunological testing was conducted according to Example 15 and results are shown in Figure 23. Figure 23 shows the response at four points in time: pre-bleed, post-prime, post- boost 1 , and post-boost 2. Anti-PnPs 6A responses are showin in Figure 23A, and anti-PnPs 19A responses are shown in Figure 23B.

Claims

1. A pharmaceutical composition, comprising:
a polymeric core particle;
a protamine protein and antigenic polysaccharide coating the particle; and
a cross-linker coupling the protamine protein.
2. The pharmaceutical composition of claim 1 , wherein the cross-linker crosslinks the protamine protein to itself and physically entangles the antigenic polysaccharide.
3. The pharmaceutical composition of claim 1 , wherein the antigenic polysaccharide is PnPsl , PnPs6A or typhoid and the cross-linker is EDC.
4. The pharmaceutical composition of claim 1 , wherein the antigenic polysaccharide is PnPsl , PnPs5 or PnPsl 4 and the cross-linker is glutaraldehyde (GA).
5. The pharmaceutical composition of claim 1 , wherein the antigenic polysaccharide is PnPs 6A and the cross-linker is ADH.
6. The pharmaceutical composition of claim 1 , wherein the antigenic polysaccharide is selected from the group consisting of typhoid, Haemophilus influenza Type b (HiB), RSV-F, serotypes of Streptococcus pneumonia, PnPsl , PnPs2, PnPs3, PnPs4, PnPs5, PnPs6A, PnPs6B, PnPs7F, PnPs8, PnPs9N, PnPs9V, PnPsl OA, PnPsl 1A, PnPs12F, PnPs14, PnPs15B, PnPs17F, PnPsl 8C, PnPsl 9F, PnPsl 9A, PnPs20, PnPs22F, PnPs23F, and PnPs33F.
7. A method for forming a pharmaceutical composition, comprising:
providing a particle comprising a biocompatible polymer;
introducing a combination of protamine protein, active agent, and cross-linker to the particle; and
cross-linking the crosslinker, wherein the combination becomes associated with the surface of the particle.
8. The method of claim 7, wherein the protamine protein is introduced prior to the active agent and cross-linker.
9. The method of claim 7, wherein the protamine protein, active agent, and cross-linker are introduced sequentially.
10. The method of claim 7, wherein the protamine protein active agent and cross-linker are introduced simultaneously.
1 1. The method of claim 7, wherein the active agent comprises an antigenic polysaccharide and a carrier protein.
12. The method of claim 1 1 , wherein the protamine protein is introduced prior to the antigenic polysaccharide, carrier protein and cross-linker.
13. The method of claim 1 1 , wherein the protamine protein and carrier protein are introduced prior to the antigenic polysaccharide and cross-linker.
14. A pharmaceutical composition, comprising:
an antigenic polysaccharide associated with a protamine protein through a cross-linker.
15. The pharmaceutical composition of claim 14, wherein the cross-linker crosslinks the protamine protein to physically entangle the polysaccharide.
16. The pharmaceutical composition of claim 14, wherein the antigenic polysaccharide is cross- linked with the protamine protein.
17. The pharmaceutical composition of claim 14, further comprising a carrier protein or antigenic protein.
18. The pharmaceutical composition of claim 17, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is not dependent on a stoichiometric ratio and the polysaccharide and carrier protein or antigenic protein are not conjugated to one another.
19. The pharmaceutical composition of claim 18, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 0.5.
20. The pharmaceutical composition of claim 19, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 0.4.
21. The pharmaceutical composition of claim 20, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 0.33.
22. The pharmaceutical composition of claim 14, wherein the antigenic polysaccharide is selected from the group consisting of typhoid, Haemophilus influenza Type b (HiB), RSV-F, serotypes of Streptococcus pneumonia, PnPsl , PnPs2, PnPs3, PnPs4, PnPs5, PnPs6A, PnPs6B, PnPs7F, PnPs8, PnPs9N, PnPs9V, PnPslOA, PnPs1 1A, PnPs12F, PnPs14,
PnPs15B, PnPs17F, PnPs18C, PnPs19F, PnPs19A, PnPs20, PnPs22F, PnPs23F, and PnPs33F.
23. The pharmaceutical composition of claim 14, wherein the cross-linker is selected from the group consisting of glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, adipic dihydrazide and bismaleimidohexane.
24. The pharmaceutical composition of claim 14, wherein the antigenic polysaccharide is PnPsl , PnPs 6A or typhoid and the cross-linker is EDC.
25. The pharmaceutical composition of claim 14, wherein the antigenic polysaccharide is PnPs 1 , PnPs5, or PnPsl 4 and the cross-linker is glutaraldehyde (GA).
26. The pharmaceutical composition of claim 14, wherein the antigenic polysaccharide is PnPs 6A and the cross-linker is ADH.
27. A pharmaceutical composition, comprising:
a biocompatible polymer associated with a protamine protein; and
an antigenic polysaccharide associated with the protamine protein through a cross- linker.
28. The pharmaceutical composition of claim 27, wherein the biocompatible polymer is formed into a particle.
29. The pharmaceutical composition of claim 27, wherein the antigenic polysaccharide and protamine protein are coupled with a surface of the particle.
30. The pharmaceutical composition of claim 27, further comprising a carrier protein or antigenic protein.
31. The pharmaceutical composition of claim 30, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is not dependent on a stoichiometric ratio and the polysaccharide and carrier protein or antigenic protein are not conjugated to one another.
32. The pharmaceutical composition of claim 31 , wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 1 :2.
33. The pharmaceutical composition of claim 32, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 2:5.
34. The pharmaceutical composition of claim 33, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 1 :3.
35. The pharmaceutical composition of claim 27, wherein the antigenic polysaccharide is selected from the group consisting of typhoid, Haemophilus influenza Type b (HiB), RSV-F, serotypes of Streptococcus pneumonia, PnPsl , PnPs2, PnPs3, PnPs4, PnPs5, PnPs6A, PnPs6B, PnPs7F, PnPs8, PnPs9N, PnPs9V, PnPslOA, PnPs1 1A, PnPs12F, PnPs14, PnPs15B, PnPs17F, PnPs18C, PnPs19F, PnPs19A, PnPs20, PnPs22F, PnPs23F, and PnPs33F.
36. The pharmaceutical composition of claim 27, wherein the cross-linker is selected from the group consisting of glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and bismaleimidohexane.
37. The pharmaceutical composition of claim 27, wherein the antigenic polysaccharide is PnPsl , PnPs6A or typhoid and the cross-linker is EDC.
38. The pharmaceutical composition of claim 27, wherein the antigenic polysaccharide is PnPsl , PnPs5 or PnPs14 and the cross-linker is glutaraldehyde (GA).
39. The pharmaceutical composition of claim 27, wherein the antigenic polysaccharide is PnPs 6A and the cross-linker is ADH.
40. A method for forming a pharmaceutical composition, comprising:
associating a protamine protein with a surface of a biocompatible particle; and crosslinking the protamine protein on the surface of the particle.
41. The method of claim 40, wherein the biocompatible particle comprises a polymer or a protein.
42. The method of claim 40, further comprising an antigenic polysaccharide associated with the particle.
43. The method of claim 42, wherein the antigenic polysaccharide is physically entangled with the crosslinked protamine protein.
44. The method of claim 42, wherein the antigenic polysaccharide is crosslinked with the protamine protein.
45. The method of claim 40, further comprising a carrier protein or an antigenic protein.
46. The method of claim 45, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is not dependent on a stoichiometric ratio and the polysaccharide and carrier protein or antigenic protein are not conjugated to one another.
47. The method of claim 46, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 1 :2.
48. The method of claim 47, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 2:5.
49. The method of claim 48, wherein the antigenic polysaccharide to carrier protein or antigenic protein ratio is less than 1 :3.
50. The method of claim 42, wherein the antigenic polysaccharide is selected from the group consisting of typhoid, Haemophilus influenza Type b (HiB), serotypes of Streptococcus pneumonia, PnPsl , PnPs2, PnPs3, PnPs4, PnPs5, PnPs6A, PnPs6B, PnPs7F, PnPs8, PnPs9N, PnPs9V, PnPslOA, PnPs1 1A, PnPs12F, PnPs14, PnPs15B, PnPs17F, PnPs18C, PnPs19F, PnPs19A, PnPs20, PnPs22F, PnPs23F, and PnPs33F.
51. The method of claim 42, wherein the cross-linker is selected from the group consisting of glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and bismaleimidohexane.
52. The method of claim 42, wherein the antigenic polysaccharide is PnPsl , PnPs6A or typhoid and the cross-linker is EDC.
53. The method of claim 42 wherein the antigenic polysaccharide is PnPsl , PnPs5, or PnPs14 and the cross-linker is glutaraldehyde (GA).
54. The method of claim 42, wherein the antigenic polysaccharide is PnPs 6A and the cross- linker is ADH.
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