WO2018227080A1 - Bacille de calmette et guérin de mycobacterium bovis délipidé (bcg) et méthodes d'utilisation - Google Patents

Bacille de calmette et guérin de mycobacterium bovis délipidé (bcg) et méthodes d'utilisation Download PDF

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WO2018227080A1
WO2018227080A1 PCT/US2018/036649 US2018036649W WO2018227080A1 WO 2018227080 A1 WO2018227080 A1 WO 2018227080A1 US 2018036649 W US2018036649 W US 2018036649W WO 2018227080 A1 WO2018227080 A1 WO 2018227080A1
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bcg
dbcg
lung
cell wall
pgl
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PCT/US2018/036649
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Juan Ignacio MOLIVA
Jordi B. TORRELLES
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Ohio State Innovation Foundation
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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
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal

Definitions

  • Mycobacterium tuberculosis the causative agent of tuberculosis (TB) continues to cause significant morbidity and mortality around the world, and the rise of extensive-, extreme-, and total-drug resistant M.tb endangers eradication efforts (World Health Organization, 2016).
  • the only currently licensed vaccine against TB Mycobacterium bovis Bacille Calmette et Guerin (BCG), is ineffective against pulmonary TB (PTB) despite it being efficacious against other forms of mycobacterial disease such as TB meningitis and military TB. What are needed are new vaccines that are effective against pulmonary TB.
  • BCG Mycobacterium bovis Bacille Calmette et Guerin
  • TDM trehalose dimycolate
  • PDL phenolic glycolipid
  • MycB Mycoside B
  • TAG tri-acyl glycerol
  • PDMI phthiocerol dimycocerosate
  • Attenuated BCGs of any preceding aspect wherein the reduction of one or more of TDM, PGL, MycB, TAG, and PDMI on the cell wall of the BCG occurs due to exposure of a delipidating agent (such as, for example, petroleum ether).
  • a delipidating agent such as, for example, petroleum ether
  • vaccines against Mycobacterium tuberculosis comprising the attenuated BCG of any of preceding aspect.
  • Also disclosed herein are methods of inhibiting a Mycobacterium tuberculosis infection in a subject comprising administering to the subject the vaccine of any preceding aspect.
  • a delipidating agent such as, for example, petroleum ether
  • TDM trehalose dimycolate
  • PDL phenolic glycolipid
  • MycB Mycoside B
  • TAG tri-acylglycerol
  • PDMI phthiocerol dimycocerosate
  • inflammatory cytokines such as, for example, T Fa, ⁇ .1 ⁇ , IL-6, and/or IL-10
  • a delipidating agent such as, for example, petroleum ether
  • TDM trehalose dimycolate
  • PDL phenolic glycolipid
  • MycB Mycoside B
  • TAG tri-acylglycerol
  • PDMI phthiocerol dimycocerosate
  • a delipidating agent such as, for example, petroleum ether
  • TDM trehalose dimycolate
  • PDL phenolic glycolipid
  • MycB Mycoside B
  • TAG tri-acylglycerol
  • PDMI phthiocerol dimycocerosate
  • FIGS 1A, IB, 1C, ID, and IE show that petroleum ether extracts TDM, PGL, MycB, PDEVIs, and TAGs without affecting viability of BCG.
  • BCG total lipid (BCG TL) extracts were included as a reference.
  • Figure 1 A shows TDM, MycB, and PGL are highly extractable by PE.
  • FIGS. 2 A, 2B, 2C, 2D, 2E, and 2F show that delipidation of BCG significantly reduces its survival and attenuates inflammatory responses in human macrophages.
  • MDMs monolayers 2.5xl0 5 cells
  • viable BCG grey bars
  • dBCG black bars
  • Colony forming unit (CFU) assays were used to assess growth of BCG or dBCG in vitro.
  • Figure 2A shows Inoculum used to infect MDMs showed no significant differences between the two groups.
  • Figure 2B shows that human macrophages were infected with BCG or dBCG at an MOI 1 : 1 and bacterial growth was determined at the indicated intervals (2-120 h).
  • Figure 2C shows that MDMs were infected with BCG or dBCG at an MOI 10: 1 and supernatants were probed for the inflammatory cytokines T Fa, IL-6, and IL- ⁇ .
  • Figure 2D shows IL-10 levels in the supernatant of dBCG infected macrophages were also significantly reduced. No significant differences were observed for IL-12p40.
  • Figure 2E shows the levels of LDH as a measure of cytotoxicity in BCG or dBCG MOI 10: 1 infected macrophages.
  • Figure 2F shows representative pictures of MDM monolayers at 120 h not infected or infected with BCG or dBCG at MOI 10: 1; final magnification: lOOx.
  • Figures 3 A, 3B, 3C, and 3D show that delipidated BCG is quickly eliminated from the lung and is associated with little pathology of the lung.
  • C57BL/6J mice were intranasally inoculated with 5xl0 5 viable BCG (grey bar or grey circle) or dBCG (black bar or black circle) bacilli. Mice were sacrificed at 2, 7, 21, 50, and 150 DPV to assess BCG bacterial burden in the lung.
  • Figure 3C shows that at 7, 21, 50, and 150 DPV, mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. Representative images are shown.
  • Figure 3D shows areas of cell aggregation and infiltration (inflammation) in BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e.
  • Figures 4 A, 4B, 4C, and 4D show that pulmonary vaccination with delipidated BCG increases memory and effector T cell responses in the lung.
  • C57BL/6J mice were intranasally inoculated with 5xl0 5 viable BCG (grey squares) or dBCG (black circles) bacilli.
  • a group of naive mice (white circles) was included as a control to assess changes in the immune cell population due to vaccination. Mice were sacrificed at 7, 21, and 50 DPV to characterize memory T cell responses in the lung.
  • Figure 4A shows the total number of CD4+ and CD8+ T cells in the lung across time.
  • Figure 4B shows the proportion of CD4+ or CD8+ T cells displaying a naive phenotype (CD62L+CD44-).
  • Figure 4C shows the proportion of CD4+ or CD8+ effector T cells (CD62L-CD44+) increased across time, and reached statistical significance in dBCG vaccinated mice at 50 DPV.
  • Figures 5 A, 5B, 5C, and 5D show that pulmonary vaccination with dBCG reduces M.tb bacterial burden in the lung and peripheral organs of infected mice.
  • C57BL/6J mice were intranasally inoculated with PBS (vehicle; white circles) or 5xl0 5 viable BCG (grey squares) or dBCG (black circles) bacilli. Fifty days later, mice were infected with a low dose aerosol of M.tb. At 21, 60, and 150 DPI mice were euthanized to assess bacterial burden in the (5A) lung, (5B) spleen, (5C) liver, and (5D) MLN.
  • Figures 6A and 6B show that pulmonary vaccination with dBCG is associated with decreased M.tb lung pathology across time.
  • C57BL/6J mice were intranasally inoculated with PBS (vehicle) or 5xl0 5 viable BCG or dBCG bacilli. Fifty days later, mice were infected with a low dose aerosol oiM.tb.
  • Figure 6A shows that at 21, 60, and 150 DPI mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology.
  • Figure 6B shows the areas of cell aggregation and infiltration (inflammation) in vehicle (white bars), BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of
  • inflammatory foci i.e. involvement
  • Representative images at a final magnification of 20X. Pooled results from n 2 with 4-5 mice/group per time-point, mean ⁇ SEM; One way ANOVA with Tukey's post-hoc test, *p ⁇ 0.05, **p ⁇ 0.01; ns: not significant.
  • Figures 7A, 7B, 7C, and 7D show that pulmonary vaccination with dBCG boosts CD69 and IL-17A, but not IFNy, responses in the lung oiM.tb infected mice.
  • Figure 7A shows a timeline showing experimental design. C57BL/6J mice were intranasally inoculated with vehicle (white circle) or 5xl0 5 viable BCG (grey square) or dBCG (black diamond) bacilli.
  • Figure 7b shows T cell activation based on the expression of CD69 on CD4+ and CD8+ T cells at 50 DPV and at 10 and 21 DPI. CD69 was significantly increased in dBCG-vaccinated mice at 21 DPI.
  • Figure 7C shows the total number of CD4+ and CD8+ T cells staining positive for IFNy after stimulation with CD3/CD28 in the presence of monensin.
  • Figure 7D shows the total number of CD4+ and CD8+ T cells staining positive for or IL-17A after stimulation with CD3/CD28 in the presence of monensin.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • An "increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • BCG not being efficacious for pulmonary TB can lie in the route of immunization as BCG is administered intradermally, whereas M.tb is a natural airborne pathogen. This noted disparity may fail to confer optimum anti -mycobacterial immunity in the lung. To circumvent this issue research has shifted toward direct pulmonary vaccination with BCG. Evidence suggests that BCG is too pathogenic to be utilized as a direct pulmonary vaccine as it can induce significant pulmonary immunopathology. For this reason no human clinical trial has yet been implemented to evaluate the efficacy of pulmonary BCG vaccination against M.tb.
  • TDM trehalose dimycolate
  • DAG/sTAGs di- and tri-acylglycerols
  • PDMIs phthiocerol dimycocerosates
  • BCG BCG with delipidating agents (such as, for example, the organic solvent petroleum ether (PE))
  • delipidating agents such as, for example, the organic solvent petroleum ether (PE)
  • TDM trehalose dimycolate
  • PDL phenolic glycolipid
  • MycB Mycoside B
  • TAGs tri-acylglycerols
  • PDMIs phthiocerol dimycocerosates
  • the attenuated BCG (i.e., the dBCG) can comprise a reduction in one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall, wherein the reduction of one or more of TDM, PGL, MycB, TAG, and/or PDMI can independently be at least a 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent reduction.
  • Attenuated BCG wherein greater than 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 of the TDM, PGL, and/or MycB have been removed from the cell wall of the BCG by the petroleum ether treatment relative to an untreated control.
  • the reduction of TDM, PGL, and/or MycB on the cell wall can be between 60 and 95%, more prefereably between 70 and 90%.
  • Attenuated BCG i.e., dBCG
  • dBCG attenuated BCG
  • the reduction of TAG and/or PDMI on the cell wall can be between 20 and 65%, more prefereably between 25 and 50%.
  • the delipidating agent can be any solvent known to remove lipids including but not limited any organic solvent including but not limited to aliphatic hydrocarbons [such as petroleum ether, sevoflurane (a nonflammable fluorinated ether)], chloroform:methanol, ⁇ -d-octyl glucoside, hexanes:isopropanol, and n-butanol among others .
  • aliphatic hydrocarbons such as petroleum ether, sevoflurane (a nonflammable fluorinated ether)
  • chloroform:methanol ⁇ -d-octyl glucoside
  • hexanes:isopropanol hexanes:isopropanol
  • n-butanol among others.
  • the delipidating agent is selected from the group consisting of petroleum ether sevoflurane (a nonflammable fluorinated ether)], chloroform methanol, ⁇ -d-octyl glucoside,
  • delipidated BCG (dBCG) was significantly attenuated compared to conventional BCG (see Figure 2) having both reduced viability and a reduction in the amount of inflammation in infected tissue.
  • a delipidating agent such as, for example, petroleum ether
  • TDM TDM
  • PGL PGL
  • MycB TAG
  • PDMI PDMI
  • Inflammatory cytokines such as TNF , IFNy, ILip, IL-2, IL-3, IL-6, IL-7, IL-9, IL- 12, IL-17, and IL-18 can play a significant role in the tissue damage associated with an infection contributing to the bulk of the cytopathology.
  • reducing the amount of inflammation associated with the immunization represents a significant benefit to the recipient by reducing the pathological implications of receiving BCG.
  • a delipidating agent such as, for example, petroleum ether
  • dBCG delipidated BCG
  • a Mycobacterium tuberculosis infection including, but not limited to infections wherein the Mycobacterium tuberculosis infection is a pulmonary infection
  • a vaccine comprising a dBCG (for example, an attenuated BCG wherein the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG has been reduced).
  • the attenuated BCG (i.e., dBCG) vaccine can be administered intranasally.
  • immunopathology associated with intrapulmonary administration for example, intranasal, aerosolized administration, topical administration to the nares, bronchial tubes or other direct intrapulmonary administration
  • methods of increasing the amount of effector memory (CD62L-CD44+) and central memory (CD62L+CD44+) T cells that arise in a subject following a BCG vaccination comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) thereby reducing the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG and administering to the subject the dBCG.
  • a delipidating agent such as, for example, petroleum ether
  • the disclosed dBCG can be used alone in a single application, it is understood and herein contemplated that the disclosed attenuated BCG (i.e., dBCG) can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times in a prime boost scenario.
  • the priming agent and boosting agent can be different.
  • the dBCGs used for the prime and each subsequent boost can comprise a difference in the number or combination of lipids reduced on the cell wall or the amount of reduction.
  • a non- delipidated BCG can be used as a boost following a dBCG prime.
  • the disclosed attenuated BCG i.e., dBCG
  • dBCG attenuated BCG
  • dBCG or any vaccine or composition comprising said dBCG can be administered as a therapeutic immunotherapy to a subject already infected with M.tb.
  • methods of inhibiting, reducing, or treating aM.tb infection comprising administering to a subject one or more of the dBCG vaccines disclosed herein.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, intranasally, including topical intranasal administration, intranasal spray, or
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 43.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation.
  • receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly, intradermally, or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions may also include one or more active ingredients such as
  • antimicrobial agents antimicrobial agents, anti -inflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the use of the delipidated BCG disclosed herein allows for a more effective inoculation, but also allows for a greater amount of bacteria to be administered to a subject without risk of disease or complications irrespective of administration route.
  • the intranasal dosage can be 10 3 , 10 4 , 2xl0 4 , 3xl0 4 , 4xl0 4 , 5xl0 4 , 6xl0 4 , 7xl0 4 , 8xl0 4 , 9xl0 4 , 10 5 , 2xl0 5 , 3xl0 5 , 4xl0 5 , or 5xl0 5 cfu.
  • Example 1 Selective Delipidation of Mycobacterium bovis BCG Enhances its Vaccine Potential against Mycobacterium tuberculosis Infection
  • TLC thin layer chromatography
  • PFMs phosphatidyl-wyo-inositol mannosides
  • Fig. lE,D phosphatidyl-wyo-inositol mannosides
  • ManLAM mannose-capped lipoarabinomannan
  • BCG and dBCG had an average uptake of 9.66 ⁇ 5.51 and 3.00 ⁇ 1.32 (M ⁇ SD), respectively, highlighting that non-polar lipids are important for entry and/or association with human macrophages.
  • dBCG infected macrophages had
  • Fig.3 A Despite equal inoculums (Fig.3 A), the ability of dBCG to persist in the lung was significantly reduced by day 2 post vaccination (DPV), a trend that continued for up to 150 days, with a continuous decrease in bacteria burden (Fig.3B).
  • DPV day 2 post vaccination
  • CFUs in the lung of dBCG-vaccinated mice were below accurate detection levels, whereas BCG- vaccinated mice had 2-3 logio CFUs in the lung (Fig.3B).
  • the levels of TNFa and IL-6 in the lungs were significantly decreased as early as 7 DPV and the levels of IL- ⁇ , IL-10, and IFNy displayed the same reduced trend.
  • B cell numbers increased significantly across time in both vaccination groups, though this was only significant between groups at 7 DPV.
  • the number of NK cells decreased across time, but was significantly increased in BCG-vaccinated mice relative to dBCG vaccinated mice at 50 DPV, while the percentage of ⁇ + ⁇ cells was not affected by either formulation despite overall significant increases across time.
  • BCG is the only licensed vaccine that primarily mediates protection via activation of CD4+, and to a lesser extent CD8+ T cells (Smith et al., 2012). BCG exerts its function as a vaccine against TB by generating a pool of memory T cells that respond rapidly upon infection with Mrf> (Vogelzang et al., 2014). For these reasons, the proportions of CD4+ and CD8+ T cells expressing markers associated with undifferentiated T cells (naive -CD62L+CD44), effector memory (TEM- CD62L CD44+) and central memory (TCM -CD62L + CD44 + ) in the lung of BCG- and dBCG-vaccinated mice were observed.
  • mice vaccinated with dBCG had significantly increased numbers of CD4+ and CD8+ T cells in the lung at 50 DPV (Fig.4A). No change to the proportion of naive cell in either subset across time (Fig.4B) was observed. Proportions of TEM CD4+ and CD8+ cells in the lung increased across time in BCG and dBCG vaccinated mice, with dBCG having significantly more at CD4+ TEM 50 DPV (Fig.4C).
  • TEM cells are believed to possess short lived immunological memory, while tissue-resident TCM cells, though far fewer than TEM in tissue, are longer-lived (Mueller et al., 2013).
  • dBCG induced greater proportions of CD4+ and CD8+ TCM cells in the lung compared to BCG vaccination (Fig.4D).
  • mice were randomized into vehicle (saline), BCG, or dBCG groups and vaccinated with 5xl0 5 bacteria via intranasal inoculation of the lung. 50 days post vaccination mice were infected with a low dose aerosol of M. tb. Bacterial burden was assessed in the lung, spleen, liver, and mediastinal lymph node (MLN) at 21, 60, and 150 days post infection (DPI).
  • M.tb mediastinal lymph node
  • dBCG vaccination was able to reduce tissue damage (symptom of M.tb infection progression to disease) by quantifying areas of cellular aggregation relative to the total size of the lung (Fig.6). dBCG vaccination attenuated pulmonary damage
  • the mean area of the inflammatory foci was 20.84 ⁇ 4.66% in vehicle-treated mice and 19.14 ⁇ 13.54% in BCG-vaccinated mice, whereas in dBCG-vaccinated was 8.30 ⁇ 3.04%.
  • dBCG is superior to BCG at reducing bacterial burden in the lung, and also more effective at preventing pulmonary immunopathology.
  • BCG remains the only vaccine available for the prevention of TB, yet it fails to confer long term immunity against PTB.
  • M.tb has evolved to be a pulmonary pathogen by taking advantage of the immunoprivileged status of the lung, where inflammatory responses are tightly regulated to prevent excessive damaging inflammation (Cooper, 2009).
  • Vaccination with BCG is administered into the dermal layer of the skin (Moliva et al., 2015).
  • Pulmonary immunopathology is the primary reason that BCG has not been repurposed as a pulmonary vaccine (Nuermberger et al., 2004; Tree et al., 2004) and immunopathology of M.tb infected animal models is a major correlate of morbidity and mortality (Moliva et al., 2015).
  • the dBCG pulmonary vaccine is therefore superior at reducing one of the most important processes that can lead to TB disease in animal models and presumably also in humans.
  • TDM has been associated with the ability to inhibit fusion between phospholipid vesicles such as those required for fusion of phagosomes with lysosomes (Spargo et al., 1991). Phagosome-lysosome (P-L) fusion is required for the killing of intracellular pathogens and for the subsequent presentation of foreign peptides along with MHC-class II to adaptive immune cells (Pieters, 2008).
  • TDM can also inhibit cellular energy metabolism by stimulating NADase activity lowering the levels of NAD and thereby, reducing the activity of NAD-dependent enzymes which can also affect the generation of adaptive immune responses (Fox et al., 2005). Furthermore, macrophages infected with TDM- expressed higher levels of MHC-II, CD Id, CD40, CD80, and CD86 and as a result were more capable of stimulating CD4+ T cell responses (Kan-Sutton et al., 2009). TDM can also induce apoptosis of lymphocytes in the thymus leading to atrophy, indicating TDM can also inhibit T cell development (Ozeki et al., 1997).
  • Some M. tb clinical isolates synthesize the trisaccharide form of PGL (PGL) and a clinical isolate of M. tb belonging to the East- Asian lineage (i.e. HN878) was found to be hypervirulent in animal models due to the presence of PGL on its cell wall (Reed et al., 2004).
  • the trisaccharide domain of PGL was able to inhibit Toll-like receptor 2 (TLR2)-induced NF-kB activation, and thus production of IL- ⁇ ⁇ , TNFa, IL-6, and CCL2, suggesting PGL enhances mycobacterial subversion of the immune system (Arbues et al., 2014).
  • TLR2 Toll-like receptor 2
  • the accumulation of TAGs on the mycobacterial cell surface can increase virulence (Reed et al., 2007).
  • purified MycB a monosaccharide PGL produced by all sub-strains ofM bovis (including BCG) but not by M.tb (Jarnagin et al., 1983) was incapable of stimulating IL- 1 ⁇ and IL-6 secretion from macrophages, but could induce TNFa (Geisel et al., 2005).
  • the accumulation of PGLs and TAGs can confer an adaptive advantage for growth in stressful environments. Similar to other lipids on the outer surface of M.
  • BCG mediates immunity through the development of antigen-specific memory T cells that respond rapidly following infection with M. tb (Shen et al., 2002).
  • Evidence suggests the success of BCG against meningeal- and miliary- TB is due to the rapid proliferation of memory T cells that quickly contain the infection, bypassing the delay in T cell priming in the lymph nodes that occurs in naive hosts (Kipnis et al., 2005).
  • memory T cells exist within two populations: Central memory (TCM) and effector memory (TEM). TcMare abundant within lymphoid tissue, respond faster to stimulus, have the ability to self-renew, and are long- lived.
  • TCM Central memory
  • TEM effector memory
  • TEM cells reside in larger numbers within non-lymphoid tissues, respond more slowly to stimulus, and are short-lived (Mueller et al., 2013).
  • Effector memory T (TEM) cells accumulate in the lung of mice vaccinated with conventional BCG via the subcutaneous route, but central memory T (TCM) cells do not (Henao-Tamayo et al., 2010). This phenomenon was also observed in humans (Purwar et al., 2011). Thus, if numbers of TCM cells are increased in the lung it can lead to faster immune responses to pathogens.
  • the data indicate that pulmonary vaccination with dBCG amplifies the number of effector memory CD4+ T cells, but more importantly, increases the number of central memory CD4+ and CD8+ T cells within the lung.
  • mice had higher numbers of CD4+CD69+ and CD8+CD69+ T cells in the lung 21 DPI, indicating that vaccination with dBCG does indeed accelerate T cell activation upon M.tb infection.
  • the number of T cells capable of producing IFNy and IL-17A in the lung was also assessed, as evidence suggests they are critical for M.tb containment (Gopal et al.,
  • IFNy was not mediating the additional protection against M.tb associated with dBCG vaccination
  • IL-17A responses were assessed as they have been implicated in conferring immunity to M.tb at mucosal sites (Gopal et al., 2014; Khader et al., 2007).
  • Significant increases were observed in the number of CD8+IL- 17A+ T cells in the lung of dBCG-vaccinated mice at 10 DPI, and significant increases in the number of CD4+IL-17A+ and CD8+IL-17A+ cells in the lung at 21 DPI; indicating that IL-17A responses may well be responsible for the increased protection observed in dBCG-vaccinated mice.
  • the data indicates IFNy has a limited ability to protect against M.tb, while IL-17A responses can be further manipulated to improve protection.
  • TB vaccine design should aim to develop single dose formulations. Though some TB vaccine boosters have shown promising results, implementation of large-scale vaccination programs may be difficult (Andersen and Kaufmann, 2014). Additionally, vaccines that require administration of components over a span of a few days/weeks pose significant logistical challenges. For these reasons, the design was centered on developing a single-dose vaccine that controls M.tb and importantly reduces pathology upon infection, a trade mark of progression to disease. TB vaccines that are inoculated into the lung have been shown to be more effective than percutaneous injections but toxic lipids on the BCG cell wall have prevented administration of BCG directly into the lung.
  • mice Specific- pathogen-free, female mice aged 6-8 weeks of the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME). Upon arrival, mice were supplied with sterilized water and chow ad libitum and acclimatized for at least one week prior to experimental manipulation. Mice were maintained in micro-isolator cages located in either a standard vivarium for all noninfectious studies or in a biosafety level three (ABSL-3) core facilities for all studies involving Mrf>. Mice were divided into three groups: Mock-vaccinated (vehicle), PBS treated BCG-vaccinated (BCG), or PE-treated BCG-vaccinated (dBCG). All experimental procedures were approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee.
  • M bovis BCG To delipidate M bovis BCG, freshly plated M. bovis BCG were harvested between 9- 14 days of growth into siliconated tubes (Fisher Scientific, Hampton, NH), suspended in 1ml of petroleum ether (PE) or phosphate buffered saline (PBS) and vortexed for two minutes, rested for five minutes, and then pelleted at 6000 g for five minutes. The procedure was repeated three times. The supernatants from the PE treated bacteria were collected, dried under N2, and kept at - 20°C until further analysis. Treated bacteria used for in vitro or in vivo studies were dried briefly in the biosafety cabinet to evaporate excess solvent, washed twice in PBS, and suspended in PBS prior to use. The viability of BCG and dBCG was assessed by performing serial dilutions, plating the bacteria onto Middlebrook 7H11 agar supplemented with OADC and counting colonies three to four weeks later.
  • PE petroleum ether
  • PBS phosphate buffered
  • PE-treated bacteria were further extracted with chloroform -methanol (C:M, 2:, v/v) at 37°C for 12 h.
  • C:M chloroform -methanol
  • PE and C:M extracted lipids were analyzed by thin layer chromatography (TLC) using aluminum -backed TLC plates with the following solvent systems: For TDM, mycoside B, and PGL (chloroform/methanol 95 :5, v/v); for phthiocerol dimycocerosates (PDIMs) and triglycerides (TAGs; petroleum ether/acetone 96:4, v/v); and phosphatidyl myoinositol mannosides (PIMs; chloroform/acetic acid/methanol/water 40:25 :3 :6, v/v/v/v).
  • TDM thin layer chromatography
  • mice were anesthetized 737 with an aerosolized solution containing 2-5% isoflurane.
  • a single cell suspension of BCG or dBCG containing approximately 5xl0 5 viable bacilli in 50 ⁇ was intranasally administrated evenly between the two nostrils allowing for inhalation into the lungs. Following administration, mice were held in an upright position for 15 seconds to ensure the entire inoculum was inhaled. Mice were then returned to their cage and monitored until recovery. At the indicated time post vaccination, mice were euthanized, and the lung was removed and processed for histological analysis, CFU enumeration, or lung cell isolation as described below.
  • the nebulizer compartment was filled with a suspension of M.tb calculated to deliver 40-100 viable bacteria into the lung.
  • organ homogenates were also plated onto OADC supplemented 7H1 1 media containing 2 ⁇ g/ml of 2-thiophenecarboxylic acid hydrazide (TCH; Sigma-Aldrich, St. Louis, MO) to exclude BCG growth (Flaherty et al., 2006).
  • TCH 2-thiophenecarboxylic acid hydrazide
  • the middle right lung was isolated from each individual mouse and inflated with and stored in 10% neutral buffered formalin.
  • Lung tissue was processed, sectioned at 4-5 ⁇ , and stained with hematoxylin and eosin (H&E) for light microscopy with lobe orientation designed to allow for maximum surface area of each lobe to be visualized. Sections were examined by a board-certified veterinary pathologist (G.B.) without prior knowledge of the experimental groups and graded according to severity, granuloma size and number.
  • H&E stained slides were digitized for morphometric analysis using Aperio ScanScope XT slide scanner (Leica, Buffalo Grove, IL) at 40X magnification. Immune cell infiltration and granulomatous tissue was calculated by manually outlining all foci and determining the total area of inflammation as a percentage of the total area of the lung.
  • PBMCs Peripheral blood mononuclear cells
  • RPMI 1640 Thermo Fisher Scientific, Waltham, MA
  • Macrophages were then collected and plated onto tissue culture plates and allowed to further differentiate for an additional seven days in RPMI 1640 containing 20% autologous serum.
  • Macrophages were infected with a single cell suspension of conventional (untreated) BCG or PE-treated BCG dissolved in PBS at a multiplicity of infection (MOI) 1 : 1 or 10: 1. Supernatants were collect at each time point and frozen at -80°C until further analysis. To enumerate CFUs, infected macrophage monolayers were lysed and serial dilutions were plated onto OADC supplemented 7H11 agar plates as described (Arcos et al., 2011). CFUs were counted 3-4 weeks later. Images of macrophage monolayers were obtained on an Olympus CKX41 SF2 microscope using an Olympus DP71 digital camera at a final magnification of lOOx.
  • Lung cell isolation 82. Lung cell were isolated. The lungs were cleared of blood via perfusion through the pulmonary artery with 10 ml PBS containing 50 U/ml heparin and placed into 2 ml cold complete-DMEM (c-DMEM) [DMEM (Mediatech, Herndon, VA), supplemented with 10% heat-inactivated FBS (Atlas Biologicals, Ft. Collins, CO), 1% HEPES buffer (1 M, Sigma- Aldrich), 10 ml lOOx nonessential amino acid solution (Sigma- Aldrich), 5 ml
  • Red blood cells were lysed using Gey's lysis buffer, and washed with c-DMEM. Cell suspensions were counted using trypan blue to exclude dead cells and resuspended at a working concentration in c- DMEM or fixed for flow cytometry as described below.
  • Cells were prepared for flow cytometry. Briefly, lung cell suspensions were adjusted to lxlO 7 cells/ml with FACS buffer supplemented with 0.1% sodium azide and incubated at 4°C for one hour. For intracellular staining, 2.5xl0 6 unfixed cells were first stimulated with 10 ⁇ g/ml CD3s, 1 ⁇ g/ml of CD28 and 3 ⁇ of monensin for 4h at 37°C, 5% C0 2 (Cyktor et al., 2013a). Cells were then labeled with cell surface markers, permeabilized using BD Cytofix/CytopermTM Plus and then labeled with intracellular markers as directed by manufacturer for 25 min at 4°C in the dark.
  • ⁇ ⁇ ⁇ were labeled with 25 ⁇ g/ml of specific fluorescent-labeled antibody for 30 min at 4°C in the dark followed by two washes with FACS buffer. Samples were read on a Becton Dickinson LSRII flow cytometer, and data were analyzed using FlowJo version 10 software. Lymphocytes were gated according to their forward- and side-scatter profiles, and CD4 or CD8 T cells were identified by the presence of specific, fluorescent-labeled antibody in combination with CD3s. Innate immune cells were blocked with CD16/CD32 (Fc block-BD Biosciences, East Rutherford, NJ) prior to staining. Antibodies used for phenotyping were: FITC-conjugated Gr-1 (RB6-8C5- BioLegend), FITC -conjugated CD3s (145-2C11-BD

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Abstract

L'invention concerne des compositions comprenant du BCG délipidé et des méthodes d'utilisation de celles-ci.
PCT/US2018/036649 2017-06-09 2018-06-08 Bacille de calmette et guérin de mycobacterium bovis délipidé (bcg) et méthodes d'utilisation WO2018227080A1 (fr)

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WO2011111898A1 (fr) * 2010-03-09 2011-09-15 Tae-Hyun Paik Procédé de préparation d'un squelette à paroi cellulaire mycobactérienne
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WO2011111898A1 (fr) * 2010-03-09 2011-09-15 Tae-Hyun Paik Procédé de préparation d'un squelette à paroi cellulaire mycobactérienne

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