US20130039947A1 - Novel immunogens and methods for discovery and screening thereof - Google Patents

Novel immunogens and methods for discovery and screening thereof Download PDF

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US20130039947A1
US20130039947A1 US13/634,357 US201113634357A US2013039947A1 US 20130039947 A1 US20130039947 A1 US 20130039947A1 US 201113634357 A US201113634357 A US 201113634357A US 2013039947 A1 US2013039947 A1 US 2013039947A1
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proteins
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pneumococcal
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Richard Malley
Yingjie Lu
Kristin L. Moffitt
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Childrens Medical Center Corp
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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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • 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
    • A61K2039/55544—Bacterial toxins
    • 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
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present application is generally directed to methods for identifying immunogens from organisms and pathogens, and in particular for identifying immunogens which when administered as vaccines elicit a cellular and/or humoral immune response.
  • the present application is also directed to pneumococcal T-cell immunogens, and methods and compositions thereof.
  • An aspect of the present invention provides for methods for identifying novel immunogens that when administered as vaccines elicit a cellular or humoral immunogenic response.
  • a method identified pneumococcal T-cell immunogens that elicit systemic IL-17A responses, and reduce or protect against pneumococcal colonization.
  • protective immunogens are identified by killing an organism with an organic solvent.
  • the organic solvent is removed, and the remaining materials re-hydrated in aqueous solution. This process releases various antigens in the liquid phase, which can then be harvested by centrifugation and collection of supernatants.
  • the liquid phase is further size-fractionated, or separated by preparative SDS gel or other methods, following which individual fractions are evaluated for immune stimulation. The most promising fractions are then evaluated further to identify components. Component proteins can then be evaluated in combination or singly to determine which are the most immunogenic and protective.
  • the present approach identified pneumococcal T-cell immunogens that both induce a Th17-cell response and protect mice from colonization. These proteins, including SP0862, SP1534 and SP2070, show promise as vaccine candidates against colonization and sepsis.
  • the novel pneumococcal immunogens as disclosed herein e.g., as disclosed in Table 1, are administered by mucosal immunization, and can be optionally administered with an adjuvant, to reduce subsequent pneumococcal nasal colonization.
  • One aspect of the present invention relates to a method for obtaining an immunogen or immunogens from a pathogen, e.g., a bacteria, comprising the steps of: (i) killing a pathogen culture with a solvent, e.g., an organic solvent; (ii) removing the solvent; (iii) resuspending the killed pathogen, e.g., a bacteria, in aqueous solution; (iv) removing particulates from the aqueous solution to retain immunogens in the aqueous solution.
  • the pathogen is a bacteria, virus, fungi, or parasite.
  • the immunogen is a protein, carbohydrate, lipid, nucleic acid, or small molecule derived from the pathogen
  • the method for obtaining an immunogen or immunogens from a pathogen further comprises the steps of: (v) isolating the proteins within the aqueous solution; and (vi) determining specific antibody or T-cell activity of the isolated antigens, in combination or each antigen individually (e.g., singly). In some embodiments, one can optionally determine specific antibody or T-cell activity of the isolated antigens (individually or in any combination) in the presence of an adjuvant and/or a vaccine scaffold.
  • Another aspect of the present invention relates to a method for obtaining bacterial T-cell-stimulating immunogens comprising the steps of: (i) killing a bacterial culture with a solvent, e.g., an organic solvent; (ii) removing the solvent; (iii) resuspending the killed bacteria in aqueous solution; (iv) removing particulates from the aqueous solution to retain the T-cell immunogens in the aqueous solution.
  • a method can optionally further comprise the steps of: (v) isolating the proteins within the aqueous solution; (vi) determining the Th17-cell inducing activity of the isolated proteins, in combination or each antigen individually (e.g., singly).
  • a pathogen or bacterial culture can be a culture of Streptococcus pneumoniae.
  • a solvent can be an organic solvent, for example, chloroform.
  • the solvent is not alcohol, and in some embodiments, the alcohol is not ethanol.
  • an immunogen is further prepared as a vaccine that reduces or protects a mammal against pneumococcal colonization.
  • a vaccine can further comprise at least one adjuvant, e.g., selected from the group comprising, but not limited to cholera toxin, CFA, IFA, alum and others commonly known in the art and disclosed herein.
  • a vaccine as disclosed herein can be administered to a subject mucosally.
  • a subject is a mammalian subject, e.g., a human, however other subjects are contemplated such as domestic and agricultural animals and the like.
  • a vaccine comprising at least one or a combination of immunogens of the pneumococcal proteins SP0862, SP1534, and SP2070, or functional fragments or proteins thereof having substantial identity.
  • a pharmaceutical composition for eliciting an immune response in a mammal comprising pneumococcal proteins SP0862, SP1534, and SP2070, or functional fragments or proteins thereof having substantial identity.
  • a pharmaceutical composition can further comprise an adjuvant.
  • compositions for eliciting an immune response in a mammal comprising the T-cell stimulating immunogens prepared according to the methods as disclosed herein.
  • a pharmaceutical composition can further comprise an adjuvant and/or a vaccine scaffold.
  • FIG. 1 shows data from a stimulation of splenocytes with elutions from preparative SDS gel separation.
  • the supernatant fraction (WCC sup) contains about 15% of total protein of whole cell vaccine killed by chloroform (WCC). Proteins in WCC sup were separated in a 4%-12% SDS gel and then eluted into fractions according to their mobility in the gel by a preparative SDS gel elution apparatus. Splenocytes from WCC immunized mice were stimulated with the same amount of protein from fraction 3 to 11 and their IL-17A production was measured by ELISA 3 days after stimulation.
  • FIG. 2 shows gels from the purification of individual proteins from E. coli. Proteins were cloned into competent E. coli cells using the pQE-30 plasmid; transformants were verified by sequencing. Proteins were expressed in successful transformants and pelleted. After lysing by sonication, his-tagged proteins in the cell lysis supernatant were purified over an agarose-Ni column. Eluted proteins were then desalted over a PD10 column and again purified by size exclusion gel filtration. Representative proteins are depicted on Coomassie stained SDS-PAGE gels.
  • SP435, SP516, SP862, SP946, SP1297, SP1415, SP1458, SP1538, SP1572, SP1733, SP2070 and SP2092 were selected from all of the proteins identified by Mass spectroscopy (see table 1) and clone
  • FIG. 4A-4B shows data on protection against colonization by intranasal immunization with a mixture of proteins.
  • a mixture of 4 ⁇ g/ml of each SPN0435, SPN1534 and SPN2070 (CHB mix) was used to immunize mice twice one week apart with 1 ⁇ g of cholera toxin (CT) as adjuvant.
  • CT cholera toxin
  • Mice immunized with CT alone or a whole cell pneumococcal preparation with CT (WCB) constituted negative and positive controls, respectively.
  • Blood was taken 3 weeks after second immunization.
  • FIG. 4A shows IL-17A production in vitro was determined in the blood samples incubated 6 days with pneumococcal whole-cell antigen.
  • FIG. 4B shows that mice were challenged intranasally with serotype 6B strain 0603 four weeks post-immunization, and the density of pneumococcal colonization was determined 7 days later by plating dilutions of
  • FIG. 5 shows protection against colonization by individual proteins in C57B1/6 mice. Immunization and challenge schedule was the same as in FIG. 4 .
  • NP colonization density was compared by the Mann-Whitney U test or by the Kruskal-Wallis test with Dunn's correction for multiple comparisons using PRISM. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 6 shows protection against colonization by individual proteins in outbred CD1 mice. Immunization and challenge schedule was the same as in FIG. 4 .
  • NP colonization density was compared by the Mann-Whitney U test or by the Kruskal-Wallis test with Dunn's correction for multiple comparisons using PRISM. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 7 shows data from stimulation of exposed mice with individual SP proteins and protection against colonization by individual proteins in CD1 mice. Immunization and challenge schedule was the same as prior. NP colonization density was compared by the Mann-Whitney U test or by the Kruskal-Wallis test with Dunn's correction for multiple comparisons using PRISM. *, p ⁇ 0.05; **, p ⁇ 0.01, ***, p ⁇ 0.001. 2070, 1534, 0862 are all SP gene numbers. 3CHB refers to the combination of SP 2070, SP 1534, SP 0862. WCV is the whole cell vaccine which serves as a positive control.
  • adjuvant refers to any agent or entity which increases the antigenic response by a cell or a subject to a target antigen.
  • pathogen refers to an organism or molecule that causes a disease or disorder in a subject.
  • pathogens include but are not limited to viruses, fungi, bacteria, parasites and other infectious organisms or molecules therefrom, as well as taxonomically related macroscopic organisms within the categories algae, fungi, yeast and protozoa or the like.
  • prokaryotic pathogen refers to a bacterial pathogen.
  • viral pathogen refers to a virus that causes illness or disease, such as HIV.
  • parasitic pathogen refers to a microorganism that is parasitic, residing for an extended period inside a host cell or host organism, that gains benefits from the host and at the same time causes illness or disease.
  • a parasitic pathogen can be bacteria, viruses, fungi, and parasites, and protists.
  • a functional fragment of an immunogen of protein “x” refers to a fragment of such a protein or peptide that mediates, effects or elicits a cellular and/or humoral immune response as similar to the protein or peptide from which it was derived.
  • a “fragment” of an antigen or immunogen of Table 1 as that term is used herein will be at least 15 amino acids in length, and can be, for example, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 25 amino acids or greater.
  • CTL Cytotoxic T Lymphocyte
  • Ag processed antigen
  • CMI cell mediated immunity
  • NK natural killer cells
  • T-cells antigen-specific cytotoxic T-lymphocytes
  • cytokines in response to a target antigen.
  • CMI refers to immune cells (such as T cells and lymphocytes) which bind to the surface of other cells that display the antigen (such as antigen presenting cells (APS)) and trigger a response.
  • the response can involve either other lymphocytes and/or any of the other white blood cells (leukocytes) and the release of cytokines.
  • CMI cell-mediated immunity
  • Cellular immunity protects the body by: (i) activating antigen-specific cytotoxic T-lymphocytes (CTLs) that are able to destroy body cells displaying epitopes of foreign antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; (2) activating macrophages and NK cells, enabling them to destroy intracellular pathogens; and (3) stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
  • CTLs cytotoxic T-lymphocytes
  • humoral immunity for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum)
  • cellular immunity for which the protective function of immunization was associated with cells.
  • immunode refers to any cell which can release a cytokine in response to a direct or indirect antigenic stimulation.
  • lympocytes including natural killer (NK) cells, T-cells (CD4+ and/or CD8+ cells), B-cells, macrophages and monocytes, Th cells; Th1 cells; Th2 cells; Tc cells; stromal cells; endothelial cells; leukocytes; dendritic cells; macrophages; mast cells and monocytes and any other cell which is capable of producing a cytokine molecule in response to direct or indirect antigen stimulation.
  • an immune cell is a lymphocyte, for example a T-cell lymphocyte.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • WCV pneumococcal whole cell vaccine candidate
  • GMP grade production During this process, organic solvents were explored as an alternative to ethanol killing. It was discovered, surprisingly, that WCV killed with chloroform (WCC) was 100 1000 ⁇ more potent than the ethanol-killed WCV. Additionally, when lyophilized WCC was reconstituted and spun down, the supernatant alone was highly protective in an animal colonization model. This lead to the hypothesis that the use of chloroform, which simply sublimates away during freezing and lyophilization, maintained soluble protective proteins of the WCV that were otherwise washed away after ethanol killing. The proteins in the WCC supernatant that contributed to its immunogenicity and protective capacities were then further characterized.
  • the present invention provides for methods for identifying antigenic candidates for vaccines.
  • possible protective antigens are identified by killing a pathogen (e.g., an infectious bacteria such as pneumococcus or an infectious virus such as influenza) with one or more solvents, or organic solvents.
  • a pathogen e.g., an infectious bacteria such as pneumococcus or an infectious virus such as influenza
  • suitable solvents include, among others, common solvents used in biological purification procedures, such as chloroform or trichloroethylene, TCE. Accordingly, in some embodiments, solvent is an organic solvent.
  • organic solvent is an art recognized term and generally refers to a solvent which belongs to the group of organic compounds and is generally used for the dissolution of organic materials.
  • Organic solvents include, but are not limited to, hydrocarbons, aromatic hydrocarbon, esters, ethers, halohydrocarbons, amines, amides, alkanolamides, ureas, alcohols, glycols, polyhydric alcohols, glycol ethers, glycol ether esters, and mixed solvents of two or more thereof.
  • organic solvents include, but are not limited to, without limitation, 1-butanol, 2-butanol, 2-butanone, Acetamide MEA (Witco Corporation, Greenwich, Conn.), acetone, acetonitrile, and n-methyl pyrrolidone, benzene, carbon tetrachloride, chlorobenzene, chloroform, cycloheptane, cyclohexane, cyclopentane, decane, dibutyl ether, dichlorobenzenes, dichloroethanes, 1,2-dichloroethane, dichloromethane (DCM), diethanolamine, diethylene glycol, diethylene glycol monomethyl ether, diglyme (diethylene glycol dimethyl ether), diglycerol, 1,2-dimethoxy-ethane (glyme, DME), dimethylether, dimethylsulfoxide (DMSO), dioxane, dipropylene glycol monomethyl ether, do
  • solvents which can be used include, but are not limited. to ester solvents such as methyl laurate, isopropyl laurate, isopropyl palmitate, isostearyl palmitate, methyl oleate, isopropyl oleate, butyl oleate, methyl linoleate, isobutyl linoleate, ethyl linoleate, isopropyl isostearate, methyl soybean oil, isobutyl soybean oil, methyl tallate, isobutyl tallate, di-isopropyl adipate, di-isopropyl sebacate, diethyl sebacate, propylene glycol monocaprate, trimethylolpropane tri-2-ethylhexanoate and glyceryl tri-2-ethylhexanoate; alcohol solvents such as isomyristyl alcohol, isopalmityl alcohol, isostearyl alcohol and o
  • the solvent is not an alcohol.
  • the solvent is not ethanol.
  • the organic solvent or solvents can be removed by methods known in the art to evaporate or sublime organic fractions in mixtures, e.g., by lyophilization.
  • the remaining materials are re-hydrated by adding water or other suitable aqueous solution. This process releases various antigens in the liquid phase, which can then be harvested by any methods common in the art, for example, by centrifugation or other phase separation techniques, and collection of supernatants.
  • various antigens that are not released in the liquid phase may be extracted by treatment of the remaining centrifuged portion with extraction techniques such as application of organic solvents, acids or bases, re-precipitation techniques, physical homogenization and/or separation, further fraction dispersion techniques, or other methods known in the art to fractionate and isolate components of solid centrifuged masses.
  • extraction techniques such as application of organic solvents, acids or bases, re-precipitation techniques, physical homogenization and/or separation, further fraction dispersion techniques, or other methods known in the art to fractionate and isolate components of solid centrifuged masses.
  • this method is not based on the mode of immunogenicity of the molecules identified. Therefore, the method allows for the isolation and identification of any type of microbial biological molecule that might elicit an immune response, including proteins, carbohydrates, lipids, nucleic acids, or small molecules. Any of these types of macromolecules that are not removed with the original organic solvent have the potential to serve as novel antigens.
  • This method is also agnostic to the normal organismal location of the molecule, and therefore allows for identification of antigens that are external (e.g. surface expressed, secreted, etc.), internal (e.g. cytoplasmic, organelle-associated, etc.), membrane bound, or capsular (associated with pathogen encapsulation layer).
  • the present method may be used to identify novel antigens from bacteria, viruses, fungi, and parasites.
  • the antigen discovery method of the present invention includes identifying antigens from pathogenic bacteria and viruses including Staphylococci (including MRSA), Streptococci species (including Group A and B), Brucella, Enterococci species; Listeria, Bacillus (including anthrax), Corynebacteria, Neisseria meningitidis, Neisseria gonorrheae, Moraxella, typeable or nontypeable Haemophilus, Haemophilus nontypeable, Pseudomonas aeruginosa and others, Salmonella typhi , non- typhi Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, E.
  • coli Clostridia, Bacteroides, Chlamydiaceae, Mycoplasma, Legionella, Treponemes, Borrelia, Candida or other yeast or other fungi, Plasmodium, Amoeba, herpes viruses, cytomegalovirus, Epstein-barr virus, varicella-zoster virus, influenza, adenoviruses, enteroviruses, or hemorrhagic viruses.
  • the methods as disclosed herein can be used to identify novel immunogens and antigens from viral, bacterial, parasitic, and tumor associated antigens.
  • preferred viral antigens include proteins from any virus where a cell-mediated immune response is desired.
  • the methods as disclosed herein can be used to identify immunogens and antigens from viruses such as HIV-1, HIV-2, hepatitis viruses (including hepatitis B and C), Ebola virus, West Nile virus, and herpes virus such as HSV-2, or bacterial antigens, e.g., from S. typhi and Mycobacteria (including M. tuberculosis ).
  • the methods as disclosed herein can be used to identify novel parasitic immunogens and antigens, including those from Plasmodium (including P. falciparum ).
  • An antigen can also include, for example, pathogenic peptides, toxins, toxoids, subunits thereof, or combinations thereof (e.g., tetanus, diphtheria toxoid, cholera subunit B, etc.).
  • the methods as disclosed herein can be used to identify novel immunogens and antigens associated with a pathology, for example an infectious disease or pathogen, or cancer or an immune disease such as an autoimmune disease.
  • a pathology for example an infectious disease or pathogen, or cancer or an immune disease such as an autoimmune disease.
  • the methods as disclosed herein can be used to identify novel immunogens and antigens from a whole virus or an attenuated virus, where an attenuated virus is a non-live or inactive virus.
  • Plotkin and Mortimer (1994) provide antigens which can be used to vaccinate animals or humans to induce an immune response specific for particular pathogens, as well as methods of preparing antigen, determining a suitable dose of antigen, assaying for induction of an immune response, and treating infection by a pathogen (e.g., bacterium, virus, fungus, or parasite).
  • a pathogen e.g., bacterium, virus, fungus, or parasite.
  • Target bacteria for use in the methods as disclosed herein include, but are not limited to: anthrax, campylobacter, cholera, diphtheria, enterotoxigenic E. coli, giardia, gonococcus, Helicobacter pylori (Lee and Chen, 1994), Hemophilus influenza B, Hemophilus influenzanon-typable, meningococcus, pertussis, pneumococcus, salmonella, shigella, Streptococcus B, group A Streptococcus , tetanus, Vibrio cholerae, yersinia, Staphylococcus, Pseudomonas species and Clostridia species.
  • Target viruses for use in the methods as disclosed herein include, but are not limited to: adenovirus, dengue serotypes 1 to 4 (Delenda et al., 1994; Fonseca et al., 1994; Smucny et al., 1995), ebola (setting et al., 1996), enterovirus, hepatitis serotypes A to E (Blum, 1995; Katkov, 1996;.Lieberman and Greenberg, 1996; Mast, 1996; Shafara et al., 1995; Smedila et al., 1994; U.S. Pat. Nos.
  • herpes simplex virus 1 or 2 human immunodeficiency virus (Deprez et al., 1996), influenza, Japanese equine encephalitis, measles, Norwalk, papilloma virus, parvovirus B19, polio, rabies, rotavirus, rubella, rubeola, vaccinia, vaccinia constructs containing genes coding for other antigens such as malaria antigens, varicella, and yellow fever.
  • Target parasites for use in the methods as disclosed herein include, but are not limited to: Entamoeba histolytica (Zhang et al., 1995); Plasmodium (Bathurst et al., 1993; Chang et al., 1989, 1992, 1994; Fries et al., 1992a, 1992b; Herrington et al., 1991; Khusmith et al., 1991; Malik et al., 1991; Migliorini et al., 1993; Pessi et al., 1991; Tam, 1988; Vreden et al., 1991; White et al., 1993; Wiesmueller et al., 1991), Leishmania (Frankenburg et al., 1996), Toxoplasmosis, and the Helminths.
  • the methods as disclosed herein can be used to identify antigens and immunogens used in biological warfare, e.g., ricin and anthrax, for which protection can be achieved via antibodies.
  • biological warfare e.g., ricin and anthrax
  • the methods as disclosed herein can be used to identify antigens and immunogens which are an intracellular pathogen.
  • a pathogen is a microorganism capable of causing damage to the host.
  • An intracellular pathogen is a microorganism that can gain entry into the interior of a cell, live inside host cells and cause damage to the host and/or host cells.
  • the pathogen can be phagocytosed and/or endocytosed by a host cell, followed by the pathogen's escape from the phagosome or endosome. The pathogen then resides intracellularly to evade other/subsequent host defense, such as antibodies, and to multiply. Phagocytosis by macrophages is a primary frontline host defense mechanism against pathogens.
  • the phagocytosed or engulfed pathogen is digested by the enzymes coming from the lysosomes.
  • the digested, smaller peptides derived from pathogen proteins are complexed with host cell MHC molecules and displayed extracellularly to other immune cells in the host so as to stimulate the immune system of the host to respond to that particular pathogen.
  • Intracellular pathogens include but are not limited to viruses, certain bacteria and certain protozoa.
  • tuberculosis tuberculosis, leprosy, typhoid fever, bacillary dysentery, plague, brucellosis, pneumonia, typhus; Rocky Mountain spotted fever, chlamydia, trachoma, gonorrhea, Listeriosis, scarlet/rheumatic fever, “strep” throat, hepatitis, AIDS, congenital viral infections, mononucleosis, Burkitts lymphoma and other lymphoproliferative diseases, cold sores, genital herpes, genital warts, cervical cancer, leishmaniasis, malaria, and trypanosomiasis to name but a few.
  • the methods as disclosed herein can be used to identify antigens and immunogens from a prokaryotic pathogen, e.g., a prokaryotic pathogen is a bacterium.
  • the intracellular prokaryotic pathogen includes but not limited to Myocobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia, Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus, Treponema pallidum, Haemophilus influenzae, Treponema pallidum, Klebsiella pneumoniae, Pseudomonas aeruginosa, Cryptosporidium parvum, Streptococcus pneumoniae, Bordetella pertussis,
  • the methods as disclosed herein can be used to identify antigens and immunogens from a viral pathogen, e.g., which includes but is not limited to Herpes simplex virus type-1, Herpes simplex virus type-2, HBV, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, poliovirus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B.
  • a viral pathogen e.g., which includes but is not limited to Herpes simplex virus type-1, Herpes simplex virus type-2, HBV, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7, Human herpes
  • the methods as disclosed herein can be used to identify antigens and immunogens from tumor cells, as many tumors are associated with the expression of a particular protein and/or the over-expression of certain proteins.
  • a tissue sample e.g., cancer biopsy sample can be added to the solvent.
  • prostate cancer is associated with elevated levels of protein such as Prostate Specific Antigen (PSA).
  • PSA Prostate Specific Antigen
  • Breast cancers can be associated with the expression and/or over-expression of protein such as Her-2, Muc-1, CEA, etc.
  • Tumors with tumor antigens include those epitopes which are recognized in eliciting T cell responses, including but not limited to the following: prostate cancer antigens (such as PSA, PSMA, etc.), breast cancer antigens (such as HER2/neu, mini-MUC, MUC-1, HER2 receptor, mammoglobulin, labyrinthine, SCP-1, NY-ESO-1, SSX-2, N-terminal blocked soluble cytokeratin, 43 kD human cancer antigens, PRAT, TUAN, Lb antigen, carcinoembryonic antigen, polyadenylate polymerase, p53, mdm-2, p21, CA15-3, oncoprotein 18/stathmin, and human glandular kallikrein), melanoma antigens, and the like.
  • prostate cancer antigens such as PSA, PSMA, etc.
  • breast cancer antigens such as HER2/neu, mini-MUC, MUC-1, HER2 receptor
  • the present unique method has identified novel T-cell antigens to pneumococci. Accordingly, the methods as disclosed herein can be particularly useful for antigen discovery in pathogens that require T-cell in addition to B-cell response. Therefore, pathogen targets of the present invention include those known or discovered to require T-cell or more specifically Th17 cell activity, including S. aureus, C. trichomatis, M. tuberculosis, viruses such as Herpes simplex virus, and others.
  • Example assays include those to directly measure antibody or T-cell responses, such as ELISA assays, cell sorting procedures, neutralization assays, or others known in the art. Additionally, it is potentially useful to monitor production of markers or secretions of cell-types, such as cytokines.
  • Example assays include T cell assays, such as elicitation of IL-17A from immune animals, or the monitoring of other cytokines such as IFN-gamma, IL-4, etc., that can identify those fractions to which antibodies from immune animals bind strongly.
  • the most-promising fractions may then be evaluated further to identify components, e.g., by mass spectroscopy or other techniques. Proteins can then be evaluated singly to determine which are immunogenic and protective. Since the separation isolation method of invention can be flexibly coupled to the immune system endpoints above, the method of the invention is useful to identify antigens that can be immunogenic in a variety of ways, including T-cell effector subtypes, antibody responses, or other adaptive or innate immune mechanisms.
  • the unique method has identified novel pneumococcal antigens, and demonstrates the utility of the approach to uncover novel immunogens from well-studied pathogens.
  • the effector T cell is the CD4+ TH17 cell: neutralization of IL-17A with anti IL-17A antibodies diminishes protection by the WCV and ll-17A receptor knockout mice are not protected by the WCV. In contrast, IFN-gamma or IL-4 deficient mice (which are skewed away from TH1 or TH2 responses, respectively) are fully protected (Lu et al., 4 PLoS Pathogens. e1000159 (2008)). Rats and mice immunized with the WCV are also significantly protected against pneumococcal sepsis in two pneumonia models (Malley et al., 2001).
  • a pneumococcal protein subunit vaccine would contain several antigens and/or be formulated with different or novel adjuvants, or incorporated in vaccine scaffolds, such as a fusion-conjugate (e.g., a fusion with a pneumolysoid and conjugation to a polysaccharide as proposed in Lu et al, Infection and Immunity, 2009) to improve immunogenicity and facilitate different routes of administration.
  • a fusion-conjugate e.g., a fusion with a pneumolysoid and conjugation to a polysaccharide as proposed in Lu et al, Infection and Immunity, 2009
  • composition comprising at least 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 12, or at least about 14, or at least about 16, or at least about 18, or at least about 20, or at least about 25, or any integer between about 2 and about 26 different antigens or more that 25 different antigens can be used, alone or in combination with an adjuvant and/or vaccine scaffold, such as a polysaccharide can be used.
  • an adjuvant and/or vaccine scaffold such as a polysaccharide
  • Mass spectroscopy identified multiple proteins within each band (range 13-23), but with some overlap in adjacent eluates.
  • a compilation of data from mass spectroscopy analysis was used to generate a table of over forty proteins that were contained within the stimulatory WCC supernatant eluates which are listed in Table 1). Based on clinical safety criteria such as lack of human homology and conservation across all twenty-two sequenced pneumococcal strains, this panel was narrowed to twelve proteins. These proteins were then expressed and purified in an E. coli expression system. Protein gels of the purified proteins yielded single bands ( FIG. 2 ) demonstrating successful purification.
  • Number of peptide matches refers to the number of unique peptide matches for a given protein within a sample. (Identifying >2 peptide matches confers a confident protein match). Number Homology w/ TIGR4 peptide human E.
  • coli Antigen GI number Proposed function matches Publications peptides Homology Location Stimulatory eluate 6 (3/11 and 3/18 eluates submitted) SP0499 57014092 Phosphogly-cerate 14 and 3 45% hum hom Cytosolic 46576835 kinase 2 SP1128 122278628 Phosphopyruvate 13 and 9 Ling et al.
  • Immunogenic purified proteins were next identified by determining which purified proteins elicited the highest IL-17A response from splenocytes of WCC immunized mice, thus prioritizing the antigens that would move into animal immunization models. As shown in FIG. 3 , several proteins met this criterion (SPN2070 (SEQ ID NO: 10; SEQ ID NO: 11), SPN1534 (SEQ ID NO: 28; SEQ ID NO: 29), SPN0435 (SEQ ID NO: 44) and SPN0862 (SEQ ID NO: 8; SEQ ID NO: 9)).
  • SPN516 SEQ ID NO: 43
  • SPN862 SEQ ID NO: 8 and 9
  • SPN946 SEQ ID NO: 35
  • SPN1297 SEQ ID NO: 20
  • SPN1415 SEQ ID NO: 38
  • SPN1458 SEQ ID NO: 33
  • SPN1572 SEQ ID NO: 19
  • SPN1733 SEQ ID NO: 35
  • candidate proteins were first evaluated using a combination vaccine comprising at least three antigens that were most stimulatory in the splenocyte stimulations ( FIG. 3 ).
  • a combination vaccine comprising at least three antigens that were most stimulatory in the splenocyte stimulations ( FIG. 3 ).
  • the immunogenicity of this combination vaccine comprising antigens SPN0435 (SEQ ID NO: 44), SPN1534 (SEQ ID NO: 28 and 29), and SPN 2070 (SEQ ID NO: 10 and 11), where the combination of such antigens is referred to as “CHB” was robust (see FIG. 4A ).
  • SPN1534 (SEQ ID NO: 28 and 29) and SPN0862 (SEQ ID NO: 8; SEQ ID NO: 9) conferred statistically significant reduction in colonization as compared with cholera toxin-immunized controls.
  • Animals immunized with SPN2092 (SEQ ID NO: 37) were not protected, validating the methods used to identify and predict those proteins, chosen from a larger pool of proteins that would be immunogenic and protective.
  • novel antigens described here namely SPN2070 (SEQ ID NO: 10; SEQ ID NO: 11), SPN1534 (SEQ ID NO: 28 and 29), and SPN0862 (SEQ ID NO: 8; SEQ ID NO: 9), and SPN0435 (SEQ ID NO: 44) demonstrate the utility of the method and provide new vaccine candidates. It has been previously reported that SPN2070 might be used as a vaccine antigen, however it was previously unknown that this protein could elicit a T-cell specific response. Additionally, the remaining proteins such as SPN1534, and SPN0862 and SPN0435 as well as other listed in Table 1 which have never before been described as possible antigens for a pneumococcal vaccine. Thus, the method presented here has approached a well-studied pathogenic bacteria and been able to identify proteins previously unknown to elicit immune-cell-specific antigenic response.
  • a vaccine can comprise SPN2070 antigen of SEQ ID NO: 10; SEQ ID NO: 11 or a portion or fragment thereof.
  • a vaccine can comprise a combination of at least two immunogens, such as SPN1534 and SPN0862, or a functional fragment or portion or protein with substantial identity thereof.
  • a vaccine can comprise any combination or one or more of SPN2070, SPN1534 and SPN0862 immunogens, or all three immunogens together, as disclosed in FIG. 7 .
  • a vaccine comprises a combination of at least two immunogens, such as SPN1534 and SPN0862, or functional fragments or proteins with substantial identity to SPN1534 and SPN0862 alone, or in combination with one or more adjuvants and/or vaccine scaffolds, such as a polypeptide scaffold.
  • a vaccine can comprise any combination or one or more of SPN2070, SPN1534 and SPN0862, or all three immunogens SPN2070, SPN1534 and SPN0862 together, or functional fragments or proteins with substantial identity to SPN2070, SPN1534 and SPN086 alone, or in combination with one or more adjuvants (e.g., as shown in FIG. 4B ) and/or in combination with a vaccine scaffold for a multivarient vaccine approach.
  • the vaccine can comprise one or any combination of immunogens of the proteins listed in Table 2, or their functional fragments, alone, or in combination with an adjuvant and/or a vaccine scaffold to produce a multivalent vaccine.
  • Table 2 lists the amino acid sequence identification numbers of the pneumococcal immunogens.
  • the vaccine can comprise at least one immunogen of the sequences listed in Table 2, or an immunogen which is a functional fragment or has substantial identity to an immunogen listed in Table 1 or 2.
  • homologous refers to the degree of sequence similarity between two peptides or between two optimally aligned nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. For example, it is based upon using a standard homology software in the default position, such as BLAST, version 2.2.14. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by similar amino acid residues (e.g., similar in steric and/or electronic nature such as, for example conservative amino acid substitutions), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology/similarity or identity refers to a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectively.
  • a sequence which is “unrelated” or “non-homologous” shares less than 40% identity, though preferably less than 25% identity with the sequences as disclosed herein.
  • substantially identical denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more usually at least 99% sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which can include deletions or additions which total 20 percent or less of the reference sequence over the comparison window.
  • the reference sequence can be a subset of a larger sequence.
  • similarity when used to describe a polypeptide, is determined by comparing the amino acid sequence and the conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • homologous or “homologues” are used interchangeably, and when used to describe a polynucleotide or polypeptide, indicates that two polynucleotides or polypeptides, or designated sequences thereof, when optimally aligned and compared, for example using BLAST, version 2.2.14 with default parameters for an alignment (see herein) are identical, with appropriate nucleotide insertions or deletions or amino-acid insertions or deletions, in at least 60% of the nucleotides, usually from about 75% to 99%, and more preferably at least about 98 to 99% of the nucleotides.
  • homolog or “homologous” as used herein also refers to homology with respect to structure and/or function. With respect to sequence homology, sequences are homologs if they are at least 60 at least 70%, at least 80%, at least 90%, at least 95% identical, at least 97% identical, or at least 99% identical. Determination of homologs of the genes or peptides of the present invention can be easily ascertained by the skilled artisan.
  • An embodiment of the present method comprises identifying a protein, “X” of interest, then removing the gene encoding for X from the organism, then replacing that gene with a gene encoding for a tagged version of the X protein (e.g., tagged with His, HA, OVA peptide, among others), which can be detected readily with monoclonal or polyclonal antibodies.
  • the organism is then grown, stained with an antibody that recognizes the tag (and is also fused to a fluorophore). Flow cytometry is then used to evaluate whether the antibodies are attached to the surface of the organism, in which case, the antigen can be deduced to be surface-expressed. Similar strategies using antibodies attached to magnetic beads can be used as well.
  • the organism can be evaluated in its encapsulated or unencapsulated form.
  • An antigen can be surface expressed, but hidden under the capsule, for selection of antigen purposes, it may be advantageous to select an antigen that is both surface expressed and accessible despite capsulation.
  • the identified immunogenic proteins or mixtures thereof may be used in a multivalent or individual vaccine, which can be administered in many forms (intramuscularly, subcutaneously, mucosally, transdermally). For example, combinations or permutations of the twelve pneumococcal immunogens may be more efficacious against colonization versus disease. A combination of several immunogens with both characteristics may provide a superior vaccine.
  • Immunogenic compositions may contain adjuvants.
  • cholera toxin CT was used as an adjuvant for intranasal administration, resulting in protection from pneumococcal colonization.
  • Alum is an affective adjuvant for subcutaneous injection.
  • Adjuvants are typically a heterogeneous group of substances that enhance the immunological response against an antigen that is administered simultaneously. In some instances, adjuvants are added to a vaccine to improve the immune response so that less vaccine is needed. Adjuvants serve to bring the antigen—the substance that stimulates the specific protective immune response—into contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration).
  • Adjuvants can also decrease the toxicity of certain antigens and provide solubility to some vaccine components. Almost all adjuvants used today for enhancement of the immune response against antigens are particles or form particles together with the antigen. In the book “Vaccine Design—the subunit and adjuvant approach” (Ed: Powell & Newman, Plenum Press, 1995) almost all known adjuvants are described both regarding their immunological activity and regarding their chemical characteristics. The type of adjuvants that do not form particles are a group of substances that act as immunological signal substances and that under normal conditions consist of the substances that are formed by the immune system as a consequence of the immunological activation after administration of particulate adjuvant systems.
  • Adjuvants for vaccines are well known in the art. Suitable additional adjuvants include, but are not limited to: complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyaninons, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum . Selection of an adjuvant depends on the animal subject to be vaccinated.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • saponin mineral gels such as aluminum hydroxide
  • mineral gels such as aluminum hydroxide
  • surface active substances such as lysolecithin, pluronic polyols, polyaninons, peptides, oil
  • Additional examples include, but are not limited to, monoglycerides and fatty acids (e. g. a mixture of mono-olein, oleic acid, and soybean oil); mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), Q521 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21), Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion); particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide
  • Phlei cell wall skeleton Phlei cell wall skeleton
  • AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulatory able to self organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects); endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and inert vehicles, such as gold particles.
  • endogenous human immunomodulators e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and inert vehicles, such as gold
  • the adjuvant can also be selected from the group consisting of QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.
  • alternative adjuvants can be used, such as a pharmaceutically acceptable adjuvant.
  • oils or hydrocarbon emulsion adjuvants should not be used for human vaccination.
  • an adjuvant suitable for use with humans is alum (alumina gel). Details of common adjuvants which are contemplated to be added to the vaccine comprising immunogens as disclosed in Table 1 include those discussed below:
  • CFA Complete Freund's Adjuvant
  • a mineral oil adjuvant uses a water-in-oil emulsion which is primarily oil.
  • the adjuvant of choice was complete Freund's adjuvant.
  • This adjuvant while potent immunogenically, also has had a significant history of frequently producing abscesses, granulomas and tissue sloughs. It contains paraffin oil, killed mycobacteria and mannide monoosleate. The paraffin oil is not metabolized; it is either expressed through the skin (via a granuloma or abscess) or phagocytized by macrophages. Multiple exposures to CFA will cause severe hypersensitivity reactions. Accidental exposure of personnel to CFA can result in sensitization to tuberculin.
  • IFA Incomplete Freund's Adjuvant
  • mineral oil adjuvant Composition similar to CFA but does not contain the killed mycobacteria so does not produce as severe reactions. Used for the booster immunizations following the initial injection with antigen-CFA. IFA can be used for initial injection if the antigen is strongly immunogenic.
  • Montanide ISA Incomplete Seppic Adjuvant: A mineral oil adjuvant. Uses mannide oleate as the major surfactant component. The antibody response is generally similar to that with IFA. Montanide ISA may have a lessened inflammatory response.
  • RAS Ribi Adjuvant System
  • TiterMax Another water-in-oil emulsion, this preperation combines a synthetic adjuvant and microparticulate silica with the metabolizable oil squalene.
  • the copolymer is the immunomodulator component.
  • Antigen is bound to the copolymer and presented to the immune cells in a highly concentrated form. Less toxicity than CFA.
  • TiterMax usually produces the same results as CFA.
  • Syntex Adjuvant Formulation A preformed oil-in-water emulsion. Uses a block copolymer for a surfactant. A muramyl dipeptide derivative is the immunostimulatory component. All in squalene, a metabolizable oil. SAF can bias the humoral response to IgG2a in the mouse, but is less toxic than CFA.
  • Aluminum Salt Adjuvants Most frequently used as adjuvants for vaccine antigen delivery. Generally weaker adjuvants than emulsion adjuvants. Aluminum Salt Adjuvants are best used with strongly immunogenic antigens, but result generally in mild inflammatory reactions.
  • Nitrocellulose-adsorbed antigen The nitrocellulose is basically inert, leading to almost no inflammatory response. Slow degradation of nitrocellulose paper allows prolonged release of antigen. Does not produce as dramatic an antibody response as CFA. Nitrocellulose-adsorbed antigen is good for use if only a small amount of antigen can be recovered from a gel band, e.g., for animal immunization.
  • Encapsulated or entrapped antigens Permits prolonged release of antigen over time; can also have immunostimulators in preparation for prolonged release. Preparation of encapsulated or entrapped antigens is complex.
  • ISCOMs Immune-stimulating complexes
  • Stable structures are formed which rapidly migrate to draining lymph nodes. Both cell-mediated and humoral immune responses are achieved. Low toxicity; ISCOMs can elicit significant antibody response.
  • Quil A is one example, QS-21 is another.
  • GerbuR adjuvant An aqueous phase adjuvant which uses immunostimulators in combination with zinc proline. GerbuR does not have a depot effect and has minimal inflammatory effect. GerbuR requires frequent boosting to maintain high titers.
  • GM-CSF is administered to the patient before the initial immune administration.
  • GM-CSF can be administered using a viral vector or an isolated protein in a pharmaceutical formulation.
  • Combinations of adjuvants can be used such as CM-CSF, I CAM and LFA. While a strong immune response is typically generated to infectious disease antigens, tumor associated antigens typically generate a weaker immune response.
  • immune stimulators such as described above are preferably used with them.
  • CMI assays are known in the art and described, for example, in United States Patent Application 2005/0014205, WO/1987/005400, U.S. Pat. No. 5,674,698 and commercially available kits such as IMMUNKNOW® CYLEX Immune cell function assay Product No. 4400, which are incorporated in their entirety by reference herein for use in the present invention.
  • a vaccine compositions described herein comprise a pharmaceutically acceptable carrier.
  • the vaccine composition described herein is formulated for administering to a mammal. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.
  • a vaccine compositions as described herein comprise pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic.
  • carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • depot forms are suitably used.
  • Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained release preparations.
  • sustained release compositions see U.S. Pat. Nos. 3,773,919, 3,887,699, EP 58,481A, EP 158,277A, Canadian Patent No. 1176565; U. Sidman et al., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982).
  • the proteins will usually be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml per application per patient.
  • other ingredients can be added to vaccine formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.
  • antioxidants e.g., ascorbic acid
  • polypeptides e.g., polyarginine or tripeptides
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • a vaccine composition as described herein for administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • a vaccine composition as described herein further comprises pharmaceutical excipients including, but not limited to biocompatible oils, physiological saline solutions, preservatives, carbohydrate, protein, amino acids, osmotic pressure controlling agents, carrier gases, pH-controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants, absorption promoters and anti-oxidative agents.
  • pharmaceutical excipients including, but not limited to biocompatible oils, physiological saline solutions, preservatives, carbohydrate, protein, amino acids, osmotic pressure controlling agents, carrier gases, pH-controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants, absorption promoters and anti-oxidative agents.
  • carbohydrates include soluble sugars such as hydropropyl cellulose, carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid, chitosan, alginate, glucose, xylose, galactose, fructose, maltose, saccharose, dextran, chondroitin sulfate, etc.
  • proteins include albumin, gelatin, etc.
  • amino acids include glycine, alanine, glutamic acid, arginine, lysine, and their salts.
  • the immunogens as described herein can be solubilized in water, a solvent such as methanol, or a buffer.
  • Suitable buffers include, but are not limited to, phosphate buffered saline Ca 2 + /Mg 2+ free (PBS), normal saline (150 mM NaCl in water), and Tris buffer.
  • Antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS.
  • acetate-PBS at acid pH may be used as a diluent after solubilization in dilute acetic acid.
  • Glycerol can be a suitable non-aqueous buffer for use in the present invention.
  • the immunogen as disclosed herein is not soluble per se, the immunogen can be present in the formulation in a suspension or even as an aggregate.
  • hydrophobic antigen can be solubilized in a detergent, for example a polypeptide containing a membrane-spanning domain.
  • an antigen in a detergent solution e.g., a cell membrane extract
  • liposomes then may be formed by removal of the detergent by dilution, dialysis, or column chromatography.
  • a vaccine composition is administered in combination with other therapeutic ingredients including, e.g., ⁇ -interferon, cytokines, chemotherapeutic agents, or anti-inflammatory or anti-viral agents.
  • a vaccine composition is administered in a pure or substantially pure form, but it is preferable to present it as a pharmaceutical composition, formulation or preparation.
  • Such formulation comprises polypeptides described herein together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • Other therapeutic ingredients include compounds that enhance antigen presentation, e.g., gamma interferon, cytokines, chemotherapeutic agents, or anti-inflammatory agents.
  • the formulations can conveniently be presented in unit dosage form and may be prepared by methods well known in the pharmaceutical art. For example, Plotkin and Mortimer (In ‘Vaccines’, 1994, W.B. Saunders Company; 2nd edition) describes vaccination of animals or humans to induce an immune response specific for particular pathogens, as well as methods of preparing antigen, determining a suitable dose of antigen, and assaying for induction of an immune response.
  • a vaccine composition as described herein further comprises an adjuvant, as described herein.
  • Formulations of vaccine compositions suitable for intravenous, intramuscular, intranasal, oral, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering the solution sterile.
  • physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like
  • physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like
  • physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like
  • physiologically compatible substances such as sodium chloride (e.g.,
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Formulations for an intranasal delivery are described in U.S. Pat. Nos. 5,427,782, 5,843,451 and 6,398,774, which are incorporated herein in their entirety by reference. Other means of mucosal administration are also encompassed herein.
  • the formulations of a vaccine composition as disclosed herein can also incorporate a stabilizer.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharide, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures.
  • Two or more stabilizers may be used in aqueous solutions at the appropriate concentration and/or pH.
  • the specific osmotic pressure in such aqueous solution is generally in the range of 0.1-3.0 osmoses, preferably in the range of 0.80-1.2.
  • the pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8.
  • a vaccine composition can be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • typical carriers such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • a method of immunization or vaccinating a mammal against pneumococcal infections comprises administering a vaccine composition described herein.
  • the vaccine compositions described herein can be administered intravenously, intranasally, intramuscularly, subcutaneously, infraperitoneally or orally.
  • a preferred route of administration is intranasal or by other mucosal route.
  • Vaccination can be conducted by conventional methods.
  • an immunogen as polypeptide as disclosed in Table 1 can be used in a suitable diluent such as saline or water, and optionally with complete or incomplete adjuvants.
  • the vaccine can be administered by any route appropriate for eliciting an immune response.
  • the vaccine can be administered once or at periodic intervals until an immune response is elicited.
  • Immune responses can be detected by a variety of methods known to those skilled in the art, including but not limited to, antibody production, cytotoxicity assay, proliferation assay and cytokine release assays.
  • samples of blood can be drawn from the immunized mammal, and analyzed for the presence of antibodies against the immieux protein used in the vaccination by ELISA and the titer of these antibodies can be determined by methods known in the art.
  • the precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. For example, a range of 25 ⁇ g -900 ⁇ g total immunogen protein can be administered intradermally, monthly for 3 months.
  • the attending physician will decide the amount of protein or vaccine composition to administer to particular individuals.
  • proteins or immunogen mixtures may be useful in diagnostics.
  • WCC supernatant with SDS buffer was loaded into 10 wells of a precast 4%-12% Bis/Tris SDS gel with -100 ⁇ g protein/lane and run at 200V over 30 mM in MES-SDS buffer. This gel was equilibrated in 2 mM phosphate buffer for 20 mM in fresh buffer three times to minimize SDS. The equilibrated gel was cut to size to fit the BioRad Mini Gel Eluter apparatus.
  • Proteins were transversely eluted through the thickness of the gel with 90 rnA of current for 20 mM. Eluates were collected into the elution chambers beneath the gel and harvested by vacuum apparatus. This method yielded fourteen eluates with one or two protein bands per eluate. The proteins within each eluate were visualized on silver stained SDS gel; bands within each eluate were reproducible from elution to elution; eluates were combined for further use.
  • Eluates were used as stimuli on splenocytes from C57B1/6 mice immunized with WCC to determine which eluates contained proteins capable of eliciting IL-17A production.
  • Supernatants were harvested after six days and assayed for IL-17A by ELISA (R&D Biosciences). We then submitted the predominant band or bands from the most stimulatory eluates for mass spectroscopic analysis.
  • Antigens were selected from the compiled mass spectroscopy data based on clinical safety criteria such as lack of human homology and conservation across sequenced pneumococcal strains.
  • Selected antigens were cloned into competent E. coli cells for expression using the pQE-30 plasmid vector incorporating a 6 ⁇ -histidine (his) tag.
  • Transformed E. coli colonies were sequenced for the protein of interest; transformants containing successfully cloned proteins were grown and induced for protein expression using IPTG. After overnight incubation allowing expression, transformants were spun down and pellets were lysed by sonication. The proteins of interest were purified from the lysed cell supernatant over a column using agarose-Ni beads to bind the His-tag; proteins were eluted in imidazole buffer after careful washing of the column. Protein-containing elutions were further purified over a desalting column prior to use in cellular stimulation assays.
  • each protein's immunogenicity was assessed in the splenocyte stimulation assay performed similarly to the assay described above used to identify which eluates contained stimulatory proteins.
  • Splenocytes from a separate cohort of ten WCC-immunized C57B1/6 animals were stimulated with 10 ⁇ g/ml of each of the twelve proteins.
  • the combination vaccine contained 4 ⁇ g of each protein per vaccine dose.
  • Vaccines were prepared with cholera toxin (CT) adjuvant.
  • CT cholera toxin
  • Control cohorts were immunized with WCV and CT or CT alone.
  • Three weeks following their 2nd immunization animals were bled; whole blood was stimulated with the whole cell antigen and IL-17A was measured from the supernatant to assess immunogenicity.
  • animals were challenged intranasally with a live type 6B pneumococcal strain.
  • animals were sacrificed and nasal washes were obtained and cultured to assess density of colonization.

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