WO2023240146A2 - COMPOSITIONS INCLUDING CANDIDA DUBLINIENSIS AND ALKALINIZED FUNGAL β-GLUCANS FOR PROTECTION AGAINST INFECTION-INDUCED SEPSIS - Google Patents

COMPOSITIONS INCLUDING CANDIDA DUBLINIENSIS AND ALKALINIZED FUNGAL β-GLUCANS FOR PROTECTION AGAINST INFECTION-INDUCED SEPSIS Download PDF

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WO2023240146A2
WO2023240146A2 PCT/US2023/068078 US2023068078W WO2023240146A2 WO 2023240146 A2 WO2023240146 A2 WO 2023240146A2 US 2023068078 W US2023068078 W US 2023068078W WO 2023240146 A2 WO2023240146 A2 WO 2023240146A2
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mice
spp
glucan
streptomyces
sepsis
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PCT/US2023/068078
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French (fr)
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WO2023240146A3 (en
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Mairi C. NOVERR
Amanda J. HARRIET
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The Administrators Of The Tulane Educational Fund
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof

Definitions

  • the present disclosure relates to methods for preventing or treating infection-induced sepsis (e.g., polymicrobial infection) in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P-glucan extract or live wild-type Candida dubliniensis. Kits for use in practicing the methods are also provided.
  • infection-induced sepsis e.g., polymicrobial infection
  • Lethal sepsis is a common sequela of GI perforations leading to intra-abdominal infections (IAI) if left untreated or misdiagnosed (Muresan et al., 2018; Blot et al., 2019).
  • IAI intra-abdominal infections
  • the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P-glucan extract.
  • the alkalinized fungal P-glucan extract is derived from Saccharomyces cerevisiae.
  • the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l ,3) backbone via P-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol.
  • the alkalinized fungal P-glucan extract may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
  • the alkalinized fungal P-glucan extract is obtained by treating purified P-glucan derived from a fungus with an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide.
  • the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising 0.1 M borate buffer at a pH of about 9.8.
  • the purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 to about 12 hours at room temperature.
  • the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N to about 10. ON at a pH of about 7 to about 14.
  • the purified P-glucan may be treated with the alkali solution comprising the alkali-metal hydroxide or an alkali earth-metal hydroxide for about 1 to about 3 hours at a temperature of from about 4° C to about 121° C.
  • the alkali-metal hydroxide is NaOH or KOH.
  • the alkali earth-metal hydroxide is Mg(OH) 2 or Ca(OH) 2 .
  • the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of live wild-type Candida dubliniensis.
  • the live wildtype Candida dubliniensis is Candida dubliniensis strain Wu284.
  • the live wild-type Candida dubliniensis may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
  • the infection-induced sepsis is caused by a polymicrobial infection.
  • the infection-induced sepsis may be caused by a viral infection, a fungal infection, or a bacterial infection.
  • the viral infection is caused by a virus selected from the group consisting of HIV, influenza virus, Ebola virus, chicken pox virus, Hepatitis B virus, HPV, measles virus, paramyxovirus, norovirus, rubella virus, Rous Sarcoma Virus, rabies virus, and rotavirus.
  • the bacterial infection is caused by gram-positive bacteria or gram-negative bacteria.
  • Examples of gram-negative bacteria include, but are not limited to, Enterobacter spp., Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp, and Vibrio spp.
  • gram-positive bacteria examples include, but are not limited to, Bacillus spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., Mycobacterium spp., Corynebacterium spp. and Clostridium spp.
  • the fungal infection is caused by a fungus selected from the group consisting of Candida spp., Cryptococcus spp., Coccidioides spp., Histoplasma spp., Blastomyces spp., and Pneumocystis spp.
  • kits for treating or preventing infection-induced sepsis in a subject in need thereof comprising a fungal beta-glucan, an alkalinization agent and instructions for use, wherein the fungal beta-glucan comprises a plurality of [3-( 1 ,3) side chains linked to a P- (1,3) backbone via [3-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol.
  • the instructions for use comprise instructions for alkali- treating the fungal beta-glucan prior to immunizing the subject.
  • alkalinization agents include any alkali solution described herein, such as those comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide.
  • the alkali-treated fungal beta-glucan is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral or intranasal administration.
  • FIGs. 1A-1E demonstrate that immunization with Candida dubliniensis induces protection against polymicrobial C. albicans/E. coli intra-abdominal infections (IAI) but not LPS-induced sepsis.
  • mice were challenged with C. albicans (1.75 x 10 7 CFUs) and / ⁇ / coli (4.5 x 10 6 CFUs) i.p. and monitored for (FIG. 2 A) survival and (FIG. 2B) morbidity/sepsis scoring for 10 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2 separate experiments.
  • FIGs. 3A-3B demonstrate that immunization with Candida dubliniensis induces protection against zymosan-induced sepsis.
  • FIGs. 4A-4B demonstrate the role of macrophages and Gr-1+ leukocytes in C. dubliniensis-induced protection in zymosan-induced sepsis.
  • For macrophage depletion mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge.
  • mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge.
  • mice were challenged with 700-1000 mg/kg zymosan and monitored for (FIG. 4A) survival and (FIG. 4B) morbidity/sepsis scoring following lethal challenge for 10 days.
  • Naive (unvaccinated) mice served as the negative control.
  • Graphs are cumulative data from 3 separate experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)).
  • FIGs. 5A-5B demonstrate that immunization with p-glucan preparations induces protection against lethal polymicrobial C. albicans/S. aureus IAI.
  • FIG. 5A Mice
  • mice were immunized by i.p. injection with unmodified (1 mg), modified P-glucan (Img), or whole glucan particle dispersible (WGP, 200 pg) 14 days prior to challenge.
  • mice were inoculated by i.p. injection with C. abicans (1.75 x 10 7 CFUs) and S.
  • aureus (8 x 10 7 CFUs) and monitored for survival and morbidity/sepsis scoring for 10 days.
  • Naive (unvaccinated) mice served as the negative control.
  • Graphs are representative of 2-3 separate experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test).
  • FIGs. 6A-6D demonstrate the role of macrophages and Gr-1+ leukocytes in
  • For macrophage depletion mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge.
  • Gr-1+ cell depletion mice were injected i.p.
  • FIG. 6A shows cumulative data of 3 separate experiments.
  • FIGs. 7A-7D demonstrate that immunization with p-glucan preparations induces protection against lethal polymicrobial C. albicans/E. coli IAI but not LPS-induced sepsis.
  • Mice were then challenged by i.p. injection with C. albicans (1.75 x 10 7 CFUs) and / ⁇ / coli (4.5 x 10 6 CFUs) (FIGs. 7A-7B) or LPS (10 mg/kg) (FIGs. 7C-7D) and monitored for survival (FIGs.
  • FIGs. 8A-8B show microbial burden monitoring following lethal Ca/Sa IAI challenge in unprotected versus protected mice.
  • aureus (8 x 10 7 CFUs), i.p. and monitored for 10 days. Moribund mice were euthanized at clinical endpoint (CE) and surviving mice at study endpoint (SE). In the case of each peritoneal lavage fluid (FIG. 8A) and spleens (FIG. 8B) were collected and plated for pathogen burdens in serial dilutions. Naive (unimmunized) mice served as the negative control. Graphs are representative of 3 separate experiments. Microbial burden values were log transformed then compared by one-way ANOVA with Tukey’s multiple comparisons test.
  • FIGs. 9A-9D demonstrate that short term abiotic immunization induces protection against lethal polymicrobial C. albicans/S. aureus IAI.
  • mice were inoculated by i.p. injection with C. albicans (1.75 x 10 7 CFUs) and S. aureus (8 x 10 7 CFUs) and monitored for survival (FIG. 9A) and morbidity/sepsis (FIG. 9B) scoring.
  • mice were euthanized, and spleens (FIG. 9D) and lavage fluid (FIG. 9C) were plated for pathogen burdens.
  • Naive (unimmunized) mice served as the negative control.
  • Graphs are representative of 1 experiment. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, p ⁇ 0.001; ****, P ⁇ 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)].
  • FIGs. 10A-10B demonstrate that long term d-zymosan immunization induces protection against lethal polymicrobial C. albicans/S. aureus IAI.
  • mice were inoculated by i.p. injection with C. albicans (1.75 x 10 7 CFUs) and S. aureus (8 x 10 7 CFUs) and monitored for survival (FIG. 10 A) and morbidity/sepsis (FIG. 10B) scoring for 10 days following challenge.
  • mice Age matched naive (unimmunized) mice served as the negative control. Graphs are representative of 2 experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
  • FIGs. 11A-11F demonstrate the role of MYD88, Dectin-1 and CARD9 in mod.
  • mice were inoculated by i.p. injection with C. albicans (1.75 x 10 7 CFUs) and S. aureus (8 x 10 7 CFUs) and monitored and monitored for (FIGs.
  • 11A, 11C, HE survival and (FIGs. 11B, HD, HF) morbidity/sepsis scoring following lethal challenge for 10 days.
  • Naive (unvaccinated) mice served as the negative control.
  • Graphs are representative of at least 2 separate experiments. The data is representative of 2 separate experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, p ⁇ 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
  • FIGs. 12A-12F demonstrate the role of IL-10 in Cd and Abiotic Vaccine-induced protection against lethal polymicrobial C. albicans/S. aureus IAI.
  • mice were inoculated by i.p. injection with C. albicans (1.75xl0 7 CFUs) and S. aureus (8xl0 7 CFUs) and monitored and monitored for (FIGs.
  • FIGs. 13A-13F demonstrate the role of macrophages and Gr-1+ leukocytes in abiotic immunization-induced protection against lethal polymicrobial C. albicans/S. aureus IAI.
  • For macrophage depletion mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge.
  • For Gr-1+ cell depletion mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge.
  • FIGs. 14A-14D demonstrate PRRs signaling in Cd and abiotic immunizations in HEK Blue reporter cell lines.
  • Dectin-IA (FIG. 14A), Dectin-2 (FIG. 14B), TLR2 (FIG. 14C) or TLR4 (FIG. 14D) reporter cells were incubated with either C. dubliniensis (1 : 10), d-zymosan (10 pg/mL), or modified P-glucan (10-100 pg/mL) in triplicate for 22 h at 37°C, 5% CO2. Plates were read at 620 nm on Synergy plate reader. PBS served as the negative control and Zymosan or LPS served as the positive control. Graphs are representative of 4 separate experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001 [for values significantly different from those of the control, one-way ANOVA],
  • FIGs. 16A-16C demonstrate that immunization with Candida dubliniensis induces protection against H1N1 Influenza challenge.
  • Mice were challenged with H1N1 influenza (15 PFU) via oropharyngeal aspiration and monitored for (FIG. 16A) survival, (FIG. 16B) morbidity/sepsis scoring and (FIG. 16C) average weight change for 15 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control.
  • Graphs are representative of 2 separate experiments.
  • mice were challenged with H1N1 influenza (15 PFU) via oropharyngeal aspiration and monitored for (FIGs. 17A, 17D, 17G) survival, (FIGs. 17B, 17E, 17H) morbidity/sepsis scoring and (FIGs. 17C, 17F, 171) average weight change for 15 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2 separate experiments.
  • FIGs. 19A-19C show percentages of macrophages and adaptive immune cells in C. dubliniensis-induced protection against H1N1 Influenza challenge.
  • FIGs. 20A-20K show measured Serum and Lung Cytokine Levels in Cd Immunized Mice.
  • Graphs are representative of 2 separate experiments. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001 [for values significantly different from those of the control, by one-way ANOVA], DETAILED DESCRIPTION
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self-administration and the administration by another.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • immune response refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the aforementioned cells or the liver or spleen (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, infectious pathogens etc.
  • the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.
  • prevention or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • a “sample” or “biological sample” may be a body fluid or a tissue sample isolated from a subject.
  • a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like.
  • sample may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids.
  • Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.
  • a blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leucocytes, and platelets), serum and plasma.
  • sp sp after a genus refers to a single unnamed species
  • spp spp. after a genus refers to more than one unnamed species.
  • Salmonella spp. refers to more than one species of Salmonella.
  • the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
  • the various modes of treatment or prevention of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • Beta-glucans are polymers containing a backbone of beta- 1,3 -linked and beta-l,4-D- glucose molecules with 1,6-linked side-chains. The frequency of these side-chains regulates secondary structures and biochemical properties. Beta-glucans are found in many foods, such as mushrooms, oats, rice, barley, seaweed, baker’s yeast and fungi. Glucan-containing extracts include Lentinan (from Shiitake mushroom), PSK (from Coriolus versicolor, laminarin (from seaweed), Schizophyllan, Betafectin and Maitake d-fraction. Beta- 1,3 -glucan is the component responsible for the majority of biological activities of zymosan, a commonly used leukocyte stimulant derived from the cell wall of Bakers’ yeast (Saccharomyces cerevisiae).
  • Beta-glucans Depending upon the source and method of isolation, beta-glucans have various degrees of branching and of linkages in the side chains. The frequency and hinge-structure of side chains determine its immunomodulatory effect. Beta-glucans of fungal and yeast origin are normally insoluble in water, but can be made soluble either by acid hydrolysis or derivatization by introducing charged groups like phosphate, sulfate, amine, carboxymethyl and so forth to the molecule (Seljelid R, Biosci. Rep. 6:845-851 (1986); Williams et al., Immunopharmacology 22:139-156 (1991)).
  • n is an integer from 0 to about 50
  • m is an integer from about 35 to about 2000
  • a pharmaceutical composition of the present technology will vary, depending upon the identity, size, and condition of the subject treated.
  • a pharmaceutical composition may comprise the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combination thereof.
  • the active ingredient may be present in the pharmaceutical composition in forms which are generally well known in the art.
  • dosages of the yeast beta-glucans administered to a subject will vary depending upon any number of factors, including but not limited to, the type of subject and type of cancer and disease state being treated, the age of the subject, the route of administration and the relative therapeutic index.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the gender and age of the patient being treated, and the like.
  • the alkalinized fungal P-glucan extract may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
  • the alkalinized fungal P-glucan extract is obtained by treating purified P-glucan derived from a fungus with an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide.
  • the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising 0.1 M borate buffer at a pH of about 9.8.
  • the purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 to about 12 hours at room temperature.
  • the purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.
  • the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N to about 10. ON at a pH of about 7 to about 14.
  • the purified P-glucan is treated with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N, about 0.02 N, about 0.03 N, about 0.04 N, about 0.05 N, about 0.06 N, about 0.07 N, about 0.08 N, about 0.09 N, about 0.1 N, about 0.2 N, about 0.3 N, about 0.4 N, about 0.5 N, about 0.6 N, about 0.7 N, about 0.8 N, about 0.9 N, about 1.0 N, about 1.5 N, about 2.0 N, about 2.5 N, about 3.0 N, about 3.5 N, about 4.0 N, about 4.5 N, about 5.0 N, about 5.5 N, about 6.0 N, about 6.5 N, about 7.0 N, about 7.5 N, about 8.0 N, about 8.5 N, about 9.0 N, about 9.5 N, or about 10.
  • an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydro
  • the infection-induced sepsis is caused by a polymicrobial infection.
  • the infection-induced sepsis may be caused by a viral infection, a fungal infection, or a bacterial infection.
  • the fungal infection is caused by a fungus selected from the group consisting of Candida spp., Cryptococcus spp., Coccidioides spp., Histoplasma spp., Blastomyces spp., and Pneumocystis spp.
  • infectious fungi that can be treated using the methods described herein include, without limitation, the following list of genus and particulars in each genus: genus Obsidian: Obsidian corymbifera: genus Ajellomyces: Ajellomyces capsulatus, Ajellomyces dermalilidis: genus Arthroderma: Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii: genus Aspergillus: Aspergillus flavus, Aspergillus fumigatus, Aspergillus ni ei", genus Blastomyces: Blastomyces dermalilidis genus Candida: Candida auris, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis
  • Neisseria flavescens Neisseria gonorrhoeae, Neisseria lactamica, Neisseria meningitides, Neisseria polysaccharea, Neisseria subflava, Neisseria weaveri, Neisseria zaodegmatis, Nocardia abscessus, Nocardia acidivorans, Nocardia africana, Nocardia alba, Nocardia altamirensis, Nocardia amamiensis, Nocardia anaemiae, Nocardia aobensis, Nocardia araoensis, Nocardia arthritidis, Nocardia asiatica, Nocardia asteroides, Nocardia beijingensis, Nocardia blacklockiae, Nocardia brasiliensis, Nocardia brevicatena, Nocardia caishijiensis, Nocardia carnea, Nocardia cerradoensis, Nocardia concave
  • Streptococcus gordonii Streptococcus halichoeri
  • Streptococcus henryi Streptococcus hyointestinalis, Streptococcus infantis
  • Streptococcus iniae Streptococcus intermedins
  • Streptococcus luteciae Streptococcus macacae
  • Streptococcus macedonicus Streptococcus marimammalium
  • Streptococcus massiliensis Streptococcus merionis, Streptococcus minor
  • Streptococcus mitis Streptococcus mutans
  • Streptococcus oligofermentans Streptococcus oralis, Streptococcus orisratti, Streptococcus orisuis, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus parauberis,
  • Streptomyces sp. SirexAA-E Streptomyces sp. strain ISP 5133, Streptomyces sp. strain ISP 5310, Streptomyces sp. strain ISP 5499, Streptomyces specialis, Streptomyces speibonae, Streptomyces spinoverrucosus, Streptomyces spiralis, Streptomyces stelliscabiei, Streptomyces stramineus, Streptomyces sulfonofaciens, Streptomyces sulphureus, Streptomyces synnetnatofortnans, Streptomyces tauricus, Streptomyces termitum, Streptomyces thermoalcalitolerans, Streptomyces thermocarboxydovorans, Streptomyces thermocarboxydus, Streptomyces thermocoprophilus, Streptomyces thermodiastaticus, Streptomyces thermolineatus, Strepto
  • thermoviolaceus Streptomyces thioluteus, Streptomyces tuirus, Streptomyces turgidiscabies, Streptomyces varsoviensis, Streptomyces vastus, Streptomyces vietnamensis, Streptomyces violaceorectus, Streptomyces viridiviolaceus, Streptomyces viridochromogenes, Streptomyces viridosporus, Streptomyces vitaminophilus, Streptomyces xiamenensis, Streptomyces yanglinensis, Streptomyces yatensis, Streptomyces yeochonensis, Streptomyces yerevanensis, Streptomyces yokosukanensis, Vibrio spp., Vibrio fluvialis, Vibrio metschnikovii, Vibrio parahaernolyticus, Vibrio vulnificus, Y
  • the subject is an immunocompromised subject, a pediatric subject, a geriatric subject, or an adult subject.
  • kits comprising a fungal beta-glucan (e.g., Saccharomyces cerevisiae beta-glucan), an alkalinization agent (e.g., borate buffer, an alkali- metal hydroxide, or an alkali earth-metal hydroxide as described herein) and instructions for using the same to treat or prevent infection-induced sepsis in a subject in need thereof, wherein the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l,3) backbone via p ⁇ ( 1 ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol.
  • the kits of the present technology comprise instructions for alkali-treating the fungal beta-glucan prior to immunizing the subject.
  • kits of the present technology are packed in suitable containers and labeled for preventing or treating infection-induced sepsis (e.g., polymicrobial infection) in a subject.
  • the above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution.
  • the kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume.
  • kits may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not.
  • the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle).
  • the kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, etc.
  • the kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the kits of the present technology may contain a written product on or in the kit container. In certain embodiments, the use of the reagents can be according to the methods of the present technology.
  • mice Female Swiss Webster mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc. Animals were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following I ACUC -approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
  • C. albicans strain DAY185 a prototrophic derivative of SC5314, was a gift from Aaron Mitchell (Carnegie Mellon University, Pittsburgh, PA).
  • the C. dubliniensis wild-type strain (Wu284) was kindly provided by Gary Moran (Trinity College, Dublin, Ireland).
  • Frozen stocks were maintained at -80°C and streaked onto yeast extract-peptone-dextrose (YPD) agar prior to use. A single colony was transferred to 10 ml of YPD broth, and the culture was shaken at 30°C for 12 to 18 h. The methicillin-resistant S.
  • aureus strain NRS383 used in all experiments was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) data bank. Frozen stocks were maintained at -80°C and streaked onto Trypticase soy agar (TSA) prior to use.
  • the strain ATCC 25922 is a commonly used quality control strain, particularly in antibody sensitivity assays and was originally isolated from a human clinical sample collected in Seattle and WA (1946). It is of serotype 06 and biotype 1.
  • LPS Mice were injected i.p. with a lethal challenge of 300 pg (10 mg/kg) of LPS obtained from E. coll Ol l i :B4 (Sigma Aldrich) and resuspended in sterile NaCl.
  • mice Female Swiss Webster mice, 5 to 7 weeks of age, or female C57BL/6 mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc or Jackson Laboratories, respectively. All PRR knockout mice were developed on a C57BL/6 background and obtained from Jackson Laboratories or collaborating laboratories (i.e., Dectin- 1" A , B6.129S6-Clec7 atmlGdb /J; MyD88' /_ , B6.129P2(SJL)-MyD88 tol 1Def 7J; CARDO' 7 ', B6.129- Card9 tolxlin /J; and IL-10' 7 ', B6.129P2-I110 tmlCgn /J).
  • mice were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following JACUC-approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
  • Abiotic fungal cell wall compounds Mice were injected i.p. with 1 dose (day -7) of 4 mg modified P-glucan preparations (prepared as described above) or 2 doses (day -14, day -7) of 1.2 mg d-zymosan (depleted zymosan from S. cerevisiae Invivogen, cat# tlrl-zyd) dissolved in sterile non-pyrogenic PBS prior to sepsis challenge.
  • CFU counts are expressed as the number of CFU/ml of peritoneal lavage fluid and CFU/g of spleen homogenates. Based on dilution volumes, the limit of detection for peritoneal lavage fluid and spleens are 20 and 50 CFU, respectively.
  • Sepsis Scoring Mice are monitored for survival for 10 days post-lethal challenge in three checks daily. Daily behavioral scoring is performed using a modified sepsis scoring criteria including fur aspect, activity level, posture, breathing quality and grimace signs to quantify morbidity and predict mortality in mice (Mai et al., 2018). Onset of sepsis is rapid in unimmunized mice; therefore, scoring in these groups is monitored for all mice through study or clinical endpoint but only reported up to the time that the majority of animals in the group have been euthanized to eliminate presentation of results that are not representative of the group as a whole. [00102] Cell Culture.
  • HEK-Blue PRR reporter cell lines (hDectin-1 A, cat#hkb-hdectla; mDectin-2, cat#hkb-mdect2; mTLR-2, cat#hkb-mtlr2, Invivogen; hTLR-4, cat#hkb-htlr4, Invivogen) were maintained in cell culture medium at 37°C, 5% CO2.
  • Mouse cytokines and chemokines measured include: Eotaxin, G-CSF, GM-CSF, IFN-y, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-la, MIP-ip, MIP-2, RANTES, TNF, and VEGF. All protocols were performed according to manufacturer instructions.
  • mice Female Swiss Webster mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc (Wilmington, MA). Animals were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following lACUC-approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
  • C. dubliniensis wild-type strain (Wu284) was kindly provided by Gary Moran (Trinity College, Dublin, Ireland). Frozen stocks were maintained at -80°C and streaked onto yeast extract-peptone-dextrose (YPD) agar prior to use. A single colony was transferred to 10 ml of YPD broth, and the culture was shaken at 30°C for 12 to 18 h. Prior to inoculation, cultures were washed 3 times by centrifugation in sterile PBS (pH 7.4), counted using a hemocytometer, and diluted in sterile PBS to prepare standardized inocula.
  • sterile PBS pH 7.4
  • the cells were washed with sterile PBS and the overlay medium was added [2x TMEM (Gibco) containing 2x MEM, non-essential amino acids (Gibco), 4 mM 1-glutamine (Lonza), 0.2% bovine serum albumin (BSA, A0336 Sigma-Aldrich), 2 mM HEPES (BioWhittaker), 2x antibiotic-antimycotic solution (Gibco) with equivalent volume of 37 °C tempered 1.8% agarose (AGTC Bioproducts, gelling point of 1.5% agarose ⁇ 27 °C) heat-dissolved in sterile PBS], Plates were incubated upside-down at 37°C, 5% CO2 for 72 h then fixed with 20% trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the overlay medium (with TCA) was removed, and cells were washed with PBS and permeabilized 10 min with absolute MeOH. Cells were washed 3x then stained with 1% crystal violet in 20% ethanol solution to visualize plaques. Wells with plaque counts >10 and ⁇ 200 were used to calculate virus titer as plaque forming units per milliliter.
  • liposome-encapsulated clodronate and liposome vehicle (1 mg/mouse; Encapsula NanoSciences) were injected i.p. in 200 pl 1 day prior to challenge or administered intranasally in 50pL 48h prior to and post-lethal challenge.
  • Clodronate (dichloromethylene-bisphosphonate) is encapsulated in the aqueous compartments of liposomes which have been filtered for size to remove larger particles that might be toxic to animals.
  • the liposomal solution is administered to the mice where phagocytic cells recognize the liposomes as foreign particles and phagocytose them. When internalized, the liposomes release clodronate into the cytosol, resulting in cell death. Liposomes without clodronate (the control group) exhibit no cellular toxicity.
  • mice were injected i.p. with either 200 pg rat anti-mouse Gr-1+ (Ly6G/Ly6C) or rat IgG2A isotype control antibodies (Bio-X-Cell) in 200 pl sterile non- pyrogenic PBS to systemically deplete PMNLs 48 h prior to and 2 h after challenge. Injections were given every 2 days for the duration of the study. Depletion was previously confirmed in our laboratory by flow cytometry.
  • Sepsis Scoring/Weight Monitoring Mice are monitored for survival in three checks per day for 15 days following challenge. Daily behavioral scoring is performed using a modified sepsis scoring criteria including fur aspect, activity level, posture, breathing quality and grimace signs to quantify morbidity and predict mortality in mice (Mai et al., 2018). Mice were weighed daily with values assessed as percentage of starting weight. Mice reached clinical endpoint at or greater than 30% loss of original weights.
  • RNA extraction and quantitation methods Isopropanol was added into the homogenized tissue and trizol mixture (1 :2 ratio) and incubated for 10 minutes at 4°C. Tissues were spun at 12,000 x g for 10 minutes at 4°C then supernatant was removed via pipette and discarded. RNA pellet was resuspended in 1 mL 75% EtOH, vortexed and spun for 5 minutes at 7500 x g at 4°C. Supernatant was discarded and pellet was dried in a biosafety cabinet for 5-10 minutes. Pellet was resuspended in 50 pL of RNase-free water with 0.1 mM EDTA and incubated at 60°C for 15 minutes. RNA yield was measured via nanodrop via A260/A280 ratio.
  • Lung Cell Isolation Washed lung tissue was cut into small pieces with surgical scissors then enzymatically digested for 45 min in 1 mL of RPMI with fetal bovine serum, heparin, liberase, EDTA, and DNASE 1 in a 12 well plate at 37°C with shaking. After enzymatic digestion, the contents of each well were pushed through a 70pm filter and washed with cold PBS with 10% FBS to stop the enzymatic process. Cell suspension was spun at 500x g for 5 minutes. Red blood cells were lysed using 1 mL of ACK (ammonium-chloride-potassium) lysing buffer for 5 min on ice and then washed with PBS twice. Cell suspension was resuspended in 2% paraformaldehyde and incubated on ice for 10 minutes to fix the cells.
  • ACK ammonium-chloride-potassium
  • Ly6C PerCP-Cy5.5 (RB6-8C5, cat#45-5931-80; Thermofisher), Ly6G AF700 (1 A8, cat#561236; BD Bioscience), CD24 APC (MI/69, cat#562349; BD Bioscience), CD45 APC-Cy7 (30-F11, cat# 557659; BD Bioscience), CDl lb BV605 (MI/70, cat# 563015; BD Bioscience), CDl lc PE (HL3, cat# 557401; BD Bioscience), F4/80 PE-Cy5 (BM8, cat# 15-4801-82; Thermofisher), MHC II PE-Cy7 (M5/114.15.2, cat# 107629; Biolegend
  • Milliplex Mouse 32-Plex Mouse serum and lung homogenate cytokine concentrations were determined using a multiplex immunoassay (cat# MCYTMAG-70K-PX32, Millipore) and analyzed using a BioPlex 200 (Bio-Rad, United States).
  • Mouse cytokines and chemokines measured include: Eotaxin, G-CSF, GM-CSF, fFN-y, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-la, MIP-ip, MIP-2, RANTES, TNF, and VEGF. All protocols were performed according to manufacturer instructions.
  • Example 2 C. dubliniensis induces Gr-1+ cell-mediated protection against lethal challenge with C. albicans/E. coli but not LPS
  • mice were inoculated (immunized) i.p. with Cd and challenged 14 d later by i.p. inoculation with Ca/Ec. Mice were monitored for survival and sepsis scores using the modified M-CASS system (Mai et al., 2018) over a 10-day period.
  • mice with Cd resulted in 80% survival following Ca/Ec challenge compared with no survival in unvaccinated mice (p ⁇ 0.0001) (FIG. IB).
  • Unvaccinated mice exhibited severe morbidity including ruffling of fur, squinting and hunched posture prior to humane euthanasia indicated by high sepsis scores in 24-48 h, while immunized mice exhibited low sepsis scores with a limited number of sepsis/morbidity indicators over the 10-day period (FIG. 1C).
  • FIG. 1C We next examined the efficacy of the live Cd immunization in protecting against sepsis induced by E.
  • LPS administration in mice rapidly induces high circulating inflammatory cytokine levels, which peak much earlier than infection- induced sepsis (Rittirsch et al., 2007; Chen et al., 2014). Additionally, physiological effects are dose-dependent and large doses of LPS are required to induce responses similar to septic shock in mice (van der Poll, 2012; Chen et al., 2014). Therefore, the abiotic and live immunization strategies disclosed herein protect against sepsis resulting from infection, not septic shock induced by high doses of toxin exposure.
  • Example 4 Immunization with fungal H-glucan compounds results in protection against polymicrobial Ca/Sa IAI sepsis predominantly mediated by Gr-1+ cells
  • the overall survival for d-zymosan immunized mice was slightly but not significantly lower; we observed an 80% survival for both challenge days.
  • the sepsis scores (FIG. 9B) were similar between both immunizations and on both challenge days. We observed low sepsis scores ( ⁇ 3) for each immunization group up to day 9 post-lethal challenge. These observed sepsis scores were significantly lower than the unimmunized mice (>6) who succumbed to the challenge by day 2 post-lethal challenge (p ⁇ 0.001-0.0001).
  • the immunized mice were sacrificed at study endpoint and spleens and peritoneal lavage fluid plated for pathogen burdens.
  • mice challenged 30d or 60d post-immunization survived (FIG. 10A) significantly better than the unimmunized and age matched mice (p ⁇ 0.05-0.01).
  • the immunized mice challenged 90d post-immunization had the same overall survival percentage (40%) as the 60d mice but were not significantly different than unimmunized mouse overall survival. This is due to earlier mortality (days 2-3 post-lethal challenge) in both 90d and unimmunized mouse groups.
  • the highest sepsis scores in the unimmunized mice with the majority reaching clinical end points by day 2 post-lethal challenge (FIG. 10B).
  • Example 5 Fungal H-glucan compounds induce protection against polymicrobial Ca/Ec IAI but not LPS sepsis
  • Example 6 Variable requirement for IL-10 in trained innate protection against polymicrobial Ca/Sa IAI following biotic and abiotic immunization
  • Example 7 PRR Signaling Pathways Differ in response to Biotics v.s Abiotic Immunization
  • mice immunized with biotic (live Cd) and abiotic (d-zymosan or mod. P-glucan) products exhibited differences in their requirements for Gr-1+ cells and macrophages.
  • P-glucan (10-100 pg/mL) to examine their respective PRR signaling pathways (FIG. 14).
  • Unmodified zymosan or LPS served as positive controls and PBS as a negative control for the assays.
  • Example 8 C. dubliniensis Induces Protection Against Lethal Challenge with C. albicans/S. aureus through CARD9 signaling pathways
  • mice lacking the MyD88 adaptor protein, Dectin- 1 receptor or the CARD9 adaptor protein. Mice were monitored for survival and morbidity for 10 days following lethal challenge. The in vivo data demonstrated a clear role for only the CARD9 adaptor protein in Cd immunization-mediated protection. In MyD88' /_ mice (FIG.
  • Example 9 Immunization with Modified Fungal H-Glucan Induces Protection Against Lethal Challenge with C. albicans/S. aureus Independent of CARD9, Dectin-1, or MyD88-dependent Signaling
  • mice depleted of circulating macrophages or Gr-1+ cells were administered i.p. anti-Gr-1 antibody to deplete Gr-1+ cells.
  • AMs intranasal mice clodronate liposomes.
  • Example 11 C. dubliniensis immunization improves viral clearance
  • Example 12 C. dubliniensis immunization protection involves macrophage activation and anti-inflammatory cytokines
  • lung immune cell populations were isolated from immunized and unimmunized mice at 3- and 7-days post influenza challenge and stained for analysis via flow cytometry.
  • the flow panel and gating strategy was adapted from Yu et al.
  • chemokines are involved in monocyte recruitment and likely have effects later in infection to replace lung resident macrophages damaged by inflammation (Chen et al., 2020; Fiore-Gartland et al., 2017).
  • MIG or CXCL9; approximately 550-560 pg/mL
  • a cytokine induced by IFNY signaling in the immunized mice on both days in the lungs (FIG. 20K) compared to unimmunized mice ( ⁇ 250 pg/mL; p ⁇ 0.05 and p ⁇ 0.01, respectively).
  • MIG amplifies IFNY signaling and is secreted by macrophages, monocytes and other antigen presenting cells.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

Abstract

The present technology relates to methods for preventing or treating infection-induced sepsis (e.g., polymicrobial infection) in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal β-glucan extract or live wild-type Candida dubliniensis. Kits for use in practicing the methods are also provided.

Description

COMPOSITIONS INCLUDING CANDIDA DUBLINIENSIS AND ALKALINIZED
FUNGAL p-GLUCANS FOR PROTECTION AGAINST INFECTION-INDUCED SEPSIS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/350,272, filed June 8, 2022, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure relates to methods for preventing or treating infection-induced sepsis (e.g., polymicrobial infection) in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P-glucan extract or live wild-type Candida dubliniensis. Kits for use in practicing the methods are also provided.
GOVERNMENT SUPPORT
[0003] This invention was made with government support under AU45096 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
[0005] Sepsis, a major cause of morbidity and mortality in critically ill patients, is the result of unrestrained inflammatory immune responses to pathogens that can result in organ dysfunction and failure, circulatory disruption, and eventual death. According to the CDC, at least 1.7 million adults in the U.S. develop sepsis annually and one of every three patient deaths in a hospital involves sepsis (Rhee et al., 2017). Despite its clinical importance, there are no vaccines available that target sepsis and few effective treatments or options for supportive care (Gyawali et al., 2019). The key to controlling sepsis lies in the prevention and/or suppression of uncontrolled host inflammation. Lethal sepsis is a common sequela of GI perforations leading to intra-abdominal infections (IAI) if left untreated or misdiagnosed (Muresan et al., 2018; Blot et al., 2019). [0006] Thus, there is an urgent need for safe and effective therapeutic agents that combat infection-induced sepsis in immune disadvantaged populations such as children, the elderly, and the immunocompromised.
SUMMARY OF THE PRESENT TECHNOLOGY
[0007] In one aspect, the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P-glucan extract. In some embodiments, the alkalinized fungal P-glucan extract is derived from Saccharomyces cerevisiae. Additionally or alternatively, in some embodiments, the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l ,3) backbone via P-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol. The alkalinized fungal P-glucan extract may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
[0008] In any of the preceding embodiments of the methods disclosed herein, the alkalinized fungal P-glucan extract is obtained by treating purified P-glucan derived from a fungus with an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide. In some embodiments, the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising 0.1 M borate buffer at a pH of about 9.8. The purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 to about 12 hours at room temperature.
[0009] In other embodiments, the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N to about 10. ON at a pH of about 7 to about 14. The purified P-glucan may be treated with the alkali solution comprising the alkali-metal hydroxide or an alkali earth-metal hydroxide for about 1 to about 3 hours at a temperature of from about 4° C to about 121° C. In certain embodiments, the alkali-metal hydroxide is NaOH or KOH. In other embodiments, the alkali earth-metal hydroxide is Mg(OH)2 or Ca(OH)2.
[0010] In another aspect, the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of live wild-type Candida dubliniensis. In some embodiments, the live wildtype Candida dubliniensis is Candida dubliniensis strain Wu284. The live wild-type Candida dubliniensis may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
[0011] In any and all embodiments of the methods disclosed herein, the infection-induced sepsis is caused by a polymicrobial infection. The infection-induced sepsis may be caused by a viral infection, a fungal infection, or a bacterial infection. In some embodiments, the viral infection is caused by a virus selected from the group consisting of HIV, influenza virus, Ebola virus, chicken pox virus, Hepatitis B virus, HPV, measles virus, paramyxovirus, norovirus, rubella virus, Rous Sarcoma Virus, rabies virus, and rotavirus. In certain embodiments, the bacterial infection is caused by gram-positive bacteria or gram-negative bacteria. Examples of gram-negative bacteria include, but are not limited to, Enterobacter spp., Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp, and Vibrio spp. Examples of gram-positive bacteria include, but are not limited to, Bacillus spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., Mycobacterium spp., Corynebacterium spp. and Clostridium spp. In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Candida spp., Cryptococcus spp., Coccidioides spp., Histoplasma spp., Blastomyces spp., and Pneumocystis spp.
[0012] In any and all embodiments of the methods disclosed herein, the subject is an immunocompromised subject, a pediatric subject, a geriatric subject, or an adult subject.
[0013] Also disclosed herein are kits for treating or preventing infection-induced sepsis in a subject in need thereof comprising a fungal beta-glucan, an alkalinization agent and instructions for use, wherein the fungal beta-glucan comprises a plurality of [3-( 1 ,3) side chains linked to a P- (1,3) backbone via [3-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol. In some embodiments, the instructions for use comprise instructions for alkali- treating the fungal beta-glucan prior to immunizing the subject. Examples of suitable alkalinization agents include any alkali solution described herein, such as those comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide. In certain embodiments, the alkali-treated fungal beta-glucan is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral or intranasal administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGs. 1A-1E demonstrate that immunization with Candida dubliniensis induces protection against polymicrobial C. albicans/E. coli intra-abdominal infections (IAI) but not LPS-induced sepsis. FIG. 1A: Initial studies confirmed that polymicrobial challenge of C. albicans (1.75 x 107 CFUs) and /■/ coli (4.5 x 106 CFUs) was lethal whereas monomicrobial challenge was not lethal (n=10 mice/group) (representative results of six repeats). FIGs. 1B-1E: For protection studies, mice (n = 10/group) were given 1.75 x 107 CFUs of live C. dubliniensis (strain Wu284, Morschhauser J et al. Mycoses. 42:29-32 (1999)), i.p. as an immunization followed by challenge with lethal C. albicans (1.75 x 107 CFUs) and /■/ coli (4.5 x 106 CFUs). (FIGs. 1B-1C) or lethal LPS (10 mg/kg) (FIGs. 1D-1E), i.p. and monitored for survival and morbidity/sepsis scoring following lethal challenge for 10 days. Animals receiving no primary challenge served as the negative (lethal) control. Mice were monitored and scored for 10 days post-lethal challenge. Graphs are representative of at least 3 separate experiments. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)).
[0015] FIGs. 2A-2B demonstrate the role of macrophages and Gr-1+ leukocytes in C. dubliniensis-induced protection against polymicrobial C. albicans/E. coli IAI. Mice (n = 10/group) were immunized by i.p. injection with 1.75 x 107 CFUs of live C. dubliniensis 14 days prior to lethal challenge. For macrophage depletion, mice were injected i.p. with liposome- encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge. For Gr-1+ cell depletion, mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge. Mice were challenged with C. albicans (1.75 x 107 CFUs) and /■/ coli (4.5 x 106 CFUs) i.p. and monitored for (FIG. 2 A) survival and (FIG. 2B) morbidity/sepsis scoring for 10 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)). [0016] FIGs. 3A-3B demonstrate that immunization with Candida dubliniensis induces protection against zymosan-induced sepsis. Mice (n = 10/group) were given 1.75 x 107 CFUs of live C. dubliniensis (strain Wu284), i.p. as an immunization followed by challenge with lethal 700-1000 mg/kg zymosan and monitored for (FIG. 3A) survival and (FIG. 3B) morbidity following lethal challenge for 10 days. Animals receiving no primary challenge served as the negative (lethal) control. Mice were monitored for survival and morbidity/sepsis scoring for 10 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 3 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for values significantly different from those of the unvaccinated control, by log rank Mantel-Cox (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)).
[0017] FIGs. 4A-4B demonstrate the role of macrophages and Gr-1+ leukocytes in C. dubliniensis-induced protection in zymosan-induced sepsis. Mice (n = 10/group) were immunized by i.p. injection with 1.75 x 107 CFUs of live C. dubliniensis 14 days prior to lethal challenge. For macrophage depletion, mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge. For Gr-1+ cell depletion, mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge. Mice were challenged with 700-1000 mg/kg zymosan and monitored for (FIG. 4A) survival and (FIG. 4B) morbidity/sepsis scoring following lethal challenge for 10 days. Naive (unvaccinated) mice served as the negative control. Graphs are cumulative data from 3 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)).
[0018] FIGs. 5A-5B demonstrate that immunization with p-glucan preparations induces protection against lethal polymicrobial C. albicans/S. aureus IAI. FIG. 5A: Mice
(n = 10/group) were immunized by i.p. injection with unmodified (1 mg), modified P-glucan (Img), or whole glucan particle dispersible (WGP, 200 pg) 14 days prior to challenge. FIG. 5B: Mice (n=10/group) were immunized by i.p. injection with 1 dose (day -14) or 2 doses (day -14, - 7) of d-zymosan (1.2 mg) prior to challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. abicans (1.75 x 107 CFUs) and S. aureus (8 x 107 CFUs) and monitored for survival and morbidity/sepsis scoring for 10 days. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2-3 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test).
[0019] FIGs. 6A-6D demonstrate the role of macrophages and Gr-1+ leukocytes in |J- glucan-induced protection against lethal polymicrobial C. albicans/S. aureus IAI. Mice (n= 10/group) were immunized with either modified P-glucan (FIGs. 6A-6B) or d-zymosan (FIGs. 6C-6D) prior to lethal challenge as described in FIG. 5. For macrophage depletion, mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge. For Gr-1+ cell depletion, mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge. Mice were challenged with 700-1000 mg/kg zymosan and monitored for (FIGs. 6A, 6C) survival and (FIGs. 6B, 6D) morbidity/sepsis scoring following lethal challenge for 10 days. Naive (unvaccinated) mice served as the negative control. Graphs are representative of at least 3 separate experiments. The exception is (FIG. 6A) that shows cumulative data of 3 separate experiments. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)).
[0020] FIGs. 7A-7D demonstrate that immunization with p-glucan preparations induces protection against lethal polymicrobial C. albicans/E. coli IAI but not LPS-induced sepsis. Mice (n= 10/group) were immunized with either modified P-glucan or d-zymosan prior to lethal challenge as described in FIG. 5. Mice were then challenged by i.p. injection with C. albicans (1.75 x 107 CFUs) and /■/ coli (4.5 x 106 CFUs) (FIGs. 7A-7B) or LPS (10 mg/kg) (FIGs. 7C-7D) and monitored for survival (FIGs. 7A, 7C) and morbidity/sepsis scoring (FIGs. 7B, 7D) for 7-10 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2-4 separate experiments and (A&B) a cumulative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)).
[0021] FIGs. 8A-8B show microbial burden monitoring following lethal Ca/Sa IAI challenge in unprotected versus protected mice. Mice (n = 5/group) previously immunized i.p. with d-Zymosan (1.2 mg) or mod. P-glucan (4 mg) then injected i.p. with liposome- encapsulated clodronate (resulting in depletion of resident peritoneal macrophages) or liposomes only 1 day prior to lethal challenge. Animals receiving no primary challenge served as the negative (lethal) control. All mice were given lethal challenge consisting of C. albicans (1.75 x 107 CFUs) and S. aureus (8 x 107 CFUs), i.p. and monitored for 10 days. Moribund mice were euthanized at clinical endpoint (CE) and surviving mice at study endpoint (SE). In the case of each peritoneal lavage fluid (FIG. 8A) and spleens (FIG. 8B) were collected and plated for pathogen burdens in serial dilutions. Naive (unimmunized) mice served as the negative control. Graphs are representative of 3 separate experiments. Microbial burden values were log transformed then compared by one-way ANOVA with Tukey’s multiple comparisons test.
[0022] FIGs. 9A-9D demonstrate that short term abiotic immunization induces protection against lethal polymicrobial C. albicans/S. aureus IAI. Mice (n = 5/group) were immunized by i.p. injection with modified P-glucan (4mg) or d-zymosan (1.2 mg) 1 or 3 days prior to challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. albicans (1.75 x 107 CFUs) and S. aureus (8 x 107 CFUs) and monitored for survival (FIG. 9A) and morbidity/sepsis (FIG. 9B) scoring. After 10 days, mice were euthanized, and spleens (FIG. 9D) and lavage fluid (FIG. 9C) were plated for pathogen burdens. Naive (unimmunized) mice served as the negative control. Graphs are representative of 1 experiment. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)].
[0023] FIGs. 10A-10B demonstrate that long term d-zymosan immunization induces protection against lethal polymicrobial C. albicans/S. aureus IAI. Mice (n = 5/group) were immunized by i.p. injection 2 doses of d-zymosan (1.2 mg) 30, 60, or 90 days prior to challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. albicans (1.75 x 107 CFUs) and S. aureus (8 x 107 CFUs) and monitored for survival (FIG. 10 A) and morbidity/sepsis (FIG. 10B) scoring for 10 days following challenge. Age matched naive (unimmunized) mice served as the negative control. Graphs are representative of 2 experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
[0024] FIGs. 11A-11F demonstrate the role of MYD88, Dectin-1 and CARD9 in mod. |J- glucan immunization-induced protection against lethal polymicrobial C. albicans/S. aureus IAI. MYD88'/_, Dectin- 1'/_ or CARD9'/_ mice (n= 10/group) were immunized with modified P-glucan prior to lethal challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. albicans (1.75 x 107 CFUs) and S. aureus (8 x 107 CFUs) and monitored and monitored for (FIGs. 11A, 11C, HE) survival and (FIGs. 11B, HD, HF) morbidity/sepsis scoring following lethal challenge for 10 days. Naive (unvaccinated) mice served as the negative control. Graphs are representative of at least 2 separate experiments. The data is representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
[0025] FIGs. 12A-12F demonstrate the role of IL-10 in Cd and Abiotic Vaccine-induced protection against lethal polymicrobial C. albicans/S. aureus IAI. Mice (n= 10/group) were immunized with either C. dubliniensis (FIGs. 12A-12B), d -zymosan (FIGs. 12C-12D), or modified [3-glucan (FIGs. 12E-12F) prior to lethal challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. albicans (1.75xl07 CFUs) and S. aureus (8xl07 CFUs) and monitored and monitored for (FIGs. 12A, 12C, 12E) survival and (FIGs. 12B, 12D, 12F) morbidity/sepsis scoring following lethal challenge for 10 days. Naive (unvaccinated) mice served as the negative control. The data is representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)].
[0026] FIGs. 13A-13F demonstrate the role of macrophages and Gr-1+ leukocytes in abiotic immunization-induced protection against lethal polymicrobial C. albicans/S. aureus IAI. Mice (n= 5/group) were immunized with either modified [3-glucan or d- zymosan prior to lethal challenge. For macrophage depletion, mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge. For Gr-1+ cell depletion, mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge. Mice were challenged with Ca Sa i.p. and monitored for survival following lethal challenge for 10 days. Mice reaching clinical endpoints or study endpoint were euthanized and serum was collected for multiplexed cytokine analysis. Naive (unimmunized) mice served as the negative control. Graphs are representative of 2 experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, one-way ANOVA followed by post hoc Student’s t-test],
[0027] FIGs. 14A-14D demonstrate PRRs signaling in Cd and abiotic immunizations in HEK Blue reporter cell lines. Dectin-IA (FIG. 14A), Dectin-2 (FIG. 14B), TLR2 (FIG. 14C) or TLR4 (FIG. 14D) reporter cells were incubated with either C. dubliniensis (1 : 10), d-zymosan (10 pg/mL), or modified P-glucan (10-100 pg/mL) in triplicate for 22 h at 37°C, 5% CO2. Plates were read at 620 nm on Synergy plate reader. PBS served as the negative control and Zymosan or LPS served as the positive control. Graphs are representative of 4 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, one-way ANOVA],
[0028] FIGs. 15A-15F demonstrate the role of MYD88, Dectin-1 and CARD9 in Cd immunization-induced protection against lethal polymicrobial C. albicans/S. aureus IAI.
MYDSS'7', Dectin- r/_ or CARD9'/_mice (n= 10/group) were immunized with either C. dubliniensis (FIGs. 15A-15B), d -zymosan (FIGs. 15C-15D), or modified P-glucan (FIGs. 15E- 15F) prior to lethal challenge. For lethal IAI challenge, mice were inoculated by i.p. injection with C. albicans (1.75xl07 CFUs) and S. aureus (8xl07 CFUs) and monitored and monitored for (FIGs. 15A, 15C, 15E) survival and (FIGs. 15B, 15D, 15F) morbidity/ sepsis scoring following lethal challenge for 10 days. Naive (unvaccinated) mice served as the negative control. Graphs are representative of at least 3 separate experiments. The data is representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
[0029] FIGs. 16A-16C demonstrate that immunization with Candida dubliniensis induces protection against H1N1 Influenza challenge. Mice (n = 10/group) were immunized by i.p. injection with 1.75xl07 CFUs of live C. dubliniensis 14 days prior to lethal challenge. Mice were challenged with H1N1 influenza (15 PFU) via oropharyngeal aspiration and monitored for (FIG. 16A) survival, (FIG. 16B) morbidity/sepsis scoring and (FIG. 16C) average weight change for 15 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, p < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t- test (sepsis scoring)].
[0030] FIGs. 17A-17I show the role of macrophages and Gr-1+ leukocytes in C dubliniensis-induced protection against H1N1 Influenza challenge. Mice (n = 10/group) were immunized by i.p. injection with 1.75xl07 CFUs of live C. dubliniensis 14 days prior to lethal challenge. For macrophage depletion, mice were injected i.p. with liposome-encapsulated clodronate or control empty liposomes 1 day prior to lethal challenge. For Gr-1+ cell depletion, mice were injected i.p. with 200 pg anti-Gr-1 or isotype control antibody 48 h prior to and 2 h after lethal challenge. Mice were challenged with H1N1 influenza (15 PFU) via oropharyngeal aspiration and monitored for (FIGs. 17A, 17D, 17G) survival, (FIGs. 17B, 17E, 17H) morbidity/sepsis scoring and (FIGs. 17C, 17F, 171) average weight change for 15 days post-lethal challenge. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, by log rank Mantel-Cox test (survival) and ANOVA followed by post hoc Student’s t-test (sepsis scoring)].
[0031] FIG. 18 shows relative expression of viral RNA in the lung tissues of H1N1- infected mice. Mice (n = 5/group) were immunized by i.p. injection with 1.75xl07 CFUs of live C. dubliniensis 14 days prior to lethal challenge. Lungs were harvested at days 3 and 7 post- lethal challenge and viral RNA copies were determined by qRT-PCR and are shown as the mean ± SEM. Naive (unvaccinated) mice served as the negative control. Graphs are representative of 1 experiment. Statistical differences between groups were analyzed using unpaired t-test.
[0032] FIGs. 19A-19C show percentages of macrophages and adaptive immune cells in C. dubliniensis-induced protection against H1N1 Influenza challenge. Mice (n = 5/group) were immunized by i.p. injection with 1.75 x 107 CFUs of live C. dubliniensis 14 days prior to lethal challenge. Lungs were harvest on days 3 and 7 post-lethal challenged and immune cells isolated fixed and stained for flow cytometry analysis. Percentages of macrophages, B cells and T cells were compared between groups. Naive (unimmunized) mice served as the negative control. Graphs are representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001 [for values significantly different from those of the control, by oneway ANOVA],
[0033] FIGs. 20A-20K show measured Serum and Lung Cytokine Levels in Cd Immunized Mice. Mice (n = 5/group) were immunized by i.p. injection with 1.75 x 107 CFUs of live C. dubliniensis 14 days prior to lethal challenge. Lungs and serum were collected on days 3 and 7 post-lethal challenged and analyzed for cytokine levels via Milliplex Mouse 32 plex. Naive (unimmunized) mice served as the negative control. Graphs are representative of 2 separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [for values significantly different from those of the control, by one-way ANOVA], DETAILED DESCRIPTION
[0034] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.
[0035] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson el al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning,' Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg el al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).
[0036] As described in the Examples herein, immunization with alkali-treated fungal P- glucans or live wild-type Candida dubliniensis conferred protection against sepsis induced by fungal, bacterial or viral pathogens. These results were remarkable because while unmodified P- glucan and whole glucan particles were ineffective in inducing protection (-30% survival at day 4 post-challenge vs 0% survival in unvaccinated mice), alkali-treated P-glucan induced strong protection with 80% survival at day 10 post-challenge (p<0.05) (FIG. 5A). Survival was dependent on Gr-1+ cells, and there was an added role for macrophages in the case of protection induced by alkali-treated P-glucan. Overall, these results demonstrate that immunization with Cd as well as abiotic fungal cell components are capable of Gr-1+ cell-mediated trained innate immune protection against sepsis of broad microbial origin.
Definitions
[0037] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
[0038] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0039] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self-administration and the administration by another.
[0040] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.
[0041] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
[0042] As used herein, “immune response” refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the aforementioned cells or the liver or spleen (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, infectious pathogens etc.
[0043] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.
[0044] As used herein, the term “overall survival” or “OS” means the observed length of life from the start of treatment to death or the date of last contact.
[0045] As used herein, “prevention” or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
[0046] As used herein, a “sample” or “biological sample” may be a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leucocytes, and platelets), serum and plasma.
[0047] As used herein, the designation “sp ” after a genus refers to a single unnamed species, while the designation “spp.” after a genus refers to more than one unnamed species. Example: Salmonella spp. refers to more than one species of Salmonella.
[0048] As used herein, "survival" refers to the subject remaining alive, and includes overall survival as well as progression free survival.
[0049] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
[0050] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
[0051] It is also to be appreciated that the various modes of treatment or prevention of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
Fungal Beta-Glucans
[0052] Beta-glucans are polymers containing a backbone of beta- 1,3 -linked and beta-l,4-D- glucose molecules with 1,6-linked side-chains. The frequency of these side-chains regulates secondary structures and biochemical properties. Beta-glucans are found in many foods, such as mushrooms, oats, rice, barley, seaweed, baker’s yeast and fungi. Glucan-containing extracts include Lentinan (from Shiitake mushroom), PSK (from Coriolus versicolor, laminarin (from seaweed), Schizophyllan, Betafectin and Maitake d-fraction. Beta- 1,3 -glucan is the component responsible for the majority of biological activities of zymosan, a commonly used leukocyte stimulant derived from the cell wall of Bakers’ yeast (Saccharomyces cerevisiae).
[0053] Depending upon the source and method of isolation, beta-glucans have various degrees of branching and of linkages in the side chains. The frequency and hinge-structure of side chains determine its immunomodulatory effect. Beta-glucans of fungal and yeast origin are normally insoluble in water, but can be made soluble either by acid hydrolysis or derivatization by introducing charged groups like phosphate, sulfate, amine, carboxymethyl and so forth to the molecule (Seljelid R, Biosci. Rep. 6:845-851 (1986); Williams et al., Immunopharmacology 22:139-156 (1991)).
[0054] The yeast beta-glucans of the present technology comprises a plurality of [3-(l ,3) side chains linked to a [3-(l ,3) backbone via [3-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol (Millipore, 346210). A generic structure of the yeast betaglucans is provided below:
Figure imgf000017_0001
[0055] An exemplar molecular structure of the yeast beta-glucans of the present technology is provided below (n is an integer from 0 to about 50, m is an integer from about 35 to about 2000):
Figure imgf000018_0001
[0056] The yeast beta-glucans of the present technology are treated with a hydrolyzing agent like an acid or enzyme to significantly reduce or eliminate (1,6) linkages within the glucan branches (a single (1,6) link is required to form the branch). In some embodiments, less than 20%, less than 10%, less than 5%, less than 3% or less than 2% of the glycosidic bonds in the beta-glucan molecule will be (1,6) linkages. These products can be particulate, semi-soluble, soluble or a gel.
[0057] Beta Glucan products having the desired structural features may be administered orally, intraperitoneally, subcutaneously, intra-muscularly or intravenously. Functional dose range of the glucans can be readily determined by one of ordinary skills in the art. For example, when administered orally the functional dose range would be in the area of 1-500 mg/kg/day, 10- 200 mg/kg/day, or 20-80 mg/kg/day. When administered parenterally, the functional dose range may be 0.1-10 mg/kg/day. In certain embodiments, the yeast beta- 1,3 -glucan is administered in the amount of 0.1-4 mg. The above mentioned pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the present technology will vary, depending upon the identity, size, and condition of the subject treated. Such a pharmaceutical composition may comprise the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combination thereof. The active ingredient may be present in the pharmaceutical composition in forms which are generally well known in the art.
[0058] Typically, dosages of the yeast beta-glucans administered to a subject, will vary depending upon any number of factors, including but not limited to, the type of subject and type of cancer and disease state being treated, the age of the subject, the route of administration and the relative therapeutic index. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the gender and age of the patient being treated, and the like.
[0059] Formulations suitable for oral administration of the yeast beta-glucans include, but are not limited to, an aqueous or oily suspension, an aqueous or oily solution, an emulsion or a particulate formulation. Such formulations can be administered by any means including, but not limited to, soft gelatin capsules.
[0060] Liquid formulations of the yeast beta-glucans disclosed herein that are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or other suitable vehicle prior to use. Administration can be by a variety of different routes including intravenous, subcutaneous, intranasal, buccal, transdermal and intrapulmonary. One of ordinary skill in the art would be able to determine the desirable routes of administration, and the kinds of formulations suitable for a particular route of administration. In general, the yeast beta-glucan can be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day.
Methods of the Present Technology
[0061] . In one aspect, the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P-glucan extract. In some embodiments, the alkalinized fungal P-glucan extract is derived from Saccharomyces cerevisiae. Additionally or alternatively, in some embodiments, the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l ,3) backbone via P-(l ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol. The alkalinized fungal P-glucan extract may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
[0062] In any of the preceding embodiments of the methods disclosed herein, the alkalinized fungal P-glucan extract is obtained by treating purified P-glucan derived from a fungus with an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide. In some embodiments, the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising 0.1 M borate buffer at a pH of about 9.8. The purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 to about 12 hours at room temperature. In certain embodiments, the purified P-glucan may be treated with the alkali solution comprising 0.1 M borate buffer for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.
[0063] Various methods for alkali-treating purified P-glucans are known in the art and are described in US 5,633,369, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N to about 10. ON at a pH of about 7 to about 14. In certain embodiments, the purified P-glucan is treated with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N, about 0.02 N, about 0.03 N, about 0.04 N, about 0.05 N, about 0.06 N, about 0.07 N, about 0.08 N, about 0.09 N, about 0.1 N, about 0.2 N, about 0.3 N, about 0.4 N, about 0.5 N, about 0.6 N, about 0.7 N, about 0.8 N, about 0.9 N, about 1.0 N, about 1.5 N, about 2.0 N, about 2.5 N, about 3.0 N, about 3.5 N, about 4.0 N, about 4.5 N, about 5.0 N, about 5.5 N, about 6.0 N, about 6.5 N, about 7.0 N, about 7.5 N, about 8.0 N, about 8.5 N, about 9.0 N, about 9.5 N, or about 10. ON at a pH of about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, or about 14. The purified P-glucan may be treated with the alkali solution comprising the alkali- metal hydroxide or an alkali earth-metal hydroxide for about 1 to about 3 hours at a temperature of from about 4° C to about 121° C. In certain embodiments, the purified P-glucan may be treated with the alkali solution comprising the alkali-metal hydroxide or an alkali earth-metal hydroxide for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours at a temperature of from about 4° C, about 5° C, about 6° C, about 7° C, about 8° C, about 9° C, about 10° C, about 11° C, about 12° C, about 13° C, about 14° C, about 15° C, about 16° C, about 17° C, about 18° C, about 19° C, about 20° C, about 21° C, about 22° C, about 23° C, about 24° C, about 25° C, about 26° C, about 27° C, about 28° C, about 29° C, about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43° C, about 44° C, about 45° C, about 46° C, about 47° C, about 48° C, about 49° C, about 50° C, about 51° C, about 52° C, about 53° C, about 54° C, about 55° C, about 56° C, about 57° C, about 58° C, about 59° C, about 60° C, about 61° C, about 62° C, about 63° C, about 64° C, about 65° C, about 66° C, about 67° C, about 68° C, about 69° C, about 70° C, about 71° C, about 72° C, about 73° C, about 74° C, about 75° C, about 76° C, about 77° C, about 78° C, about 79° C, about 80° C, about 81° C, about 82° C, about 83° C, about 84° C, about 85° C, about 86° C, about 87° C, about 88° C, about 89° C, about 90° C, about 91° C, about 92° C, about 93° C, about 94° C, about 95° C, about 96° C, about 97° C, about 98° C, about 99° C, about 100° C, about 101° C, about 102° C, about 103° C, about 104° C, about 105° C, about 106° C, about 107° C, about 108° C, about 109° C, about 110° C, about 111° C, about 112° C, about 113° C, about 114° C, about 115° C, about 116° C, about 117° C, about 118° C, about 119° C, about 120° C, or about 121° C. In certain embodiments, the alkali-metal hydroxide is NaOH or KOH. In other embodiments, the alkali earth-metal hydroxide is Mg(OH)2 or Ca(OH)2.
[0064] In another aspect, the present disclosure provides a method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of live wild-type Candida dubliniensis. In some embodiments, the live wildtype Candida dubliniensis is Candida dubliniensis strain Wu284. The live wild-type Candida dubliniensis may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
[0065] In any and all embodiments of the methods disclosed herein, the infection-induced sepsis is caused by a polymicrobial infection. The infection-induced sepsis may be caused by a viral infection, a fungal infection, or a bacterial infection.
[0066] In some embodiments, the viral infection is caused by a virus selected from the group consisting of HIV, influenza virus, Ebola virus, chicken pox virus, Hepatitis B virus, HPV, measles virus, paramyxovirus, norovirus, rubella virus, Rous Sarcoma Virus, rabies virus, and rotavirus.
[0067] In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Candida spp., Cryptococcus spp., Coccidioides spp., Histoplasma spp., Blastomyces spp., and Pneumocystis spp. Representative examples of infectious fungi that can be treated using the methods described herein include, without limitation, the following list of genus and particulars in each genus: genus Obsidian: Obsidian corymbifera: genus Ajellomyces: Ajellomyces capsulatus, Ajellomyces dermalilidis: genus Arthroderma: Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii: genus Aspergillus: Aspergillus flavus, Aspergillus fumigatus, Aspergillus ni ei", genus Blastomyces: Blastomyces dermalilidis genus Candida: Candida auris, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pelliculosa, genus Cladophialophora:
Cladophialophora carrionii: genus Coccidioides: Coccidioides immitis, Coccidioides posadasii: genus Cryptococcus: Cryptococcus neoformans, Cryptococcus gallii: genus Cunninghamella: Cunninghamella sp.; genus Epidermophyton: Epidermophyton floccosum genus Exophiala: Exophiala dermalilidis: genus Filobasidiella: Filobasidiella neoformans,' genus Fonsecaea: Fonsecaea pedrosoi,' genus Fusarium: Fusarium solani,' genus Geotrichum: Geotrichum candidum,' genus Histoplasma: Histoplasma capsulatum,' genus Hortaea: Hortaea werneckii,' genus Issatschenkia: Issatschenkia orientalis,' genus Madurella: Madurella grisae,' genus Malassezia: Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia pachydermatis, Malassezia restricta, Malassezia sloofftae, Malassezia sympodialis,' genus Microsporum: Microsporum canis, Microsporum fulvum, Microsporum gypseunr, genus Mucor: Mucor circinelloides,' genus Nectria: Nectria haematococca, genus Paecilomyces: Paecilomyces variotir, genus Paracoccidioides: Paracoccidioides brasiliensis,' genus Penicillium: Penicillium marneffei,' genus Pichia, Pichia anomala, Pichia guilliermondii,' genus Pneumocystis: Pneumocystis carinii,' genus Pseudalle scher ia: Pseudallescheria boydii,' genus Rhizopus: Rhizopus oryzae,' genus Rhodotorula: Rhodotorula rubra, genus Scedosporium: Scedosporium apiospermunr, genus Schizophyllum: Schizophyllum commune,' genus Sporothrix: Sporothrix schenckii,' genus Trichophyton: Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceunr, and of the genus Trichosporon: Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides.
[0068] In certain embodiments, the bacterial infection is caused by gram-positive bacteria or gram-negative bacteria. Examples of gram-negative bacteria include, but are not limited to, Enter obacter spp., Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp, and Vibrio spp. Examples of gram-positive bacteria include, but are not limited to, Bacillus spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., Mycobacterium spp., Corynebacterium spp. and Clostridium spp.
[0069] Representative examples of infectious fungi that can be treated using the methods described herein include, without limitation, Acinetobacter baumannii, Acinetobacter calcoaceticus, Actinobacillus arthritidis, Actinobacillus capsulatus, Actinobacillus delphinicola, Actinobacillus equuli subsp. haemolyticus, Actinobacillus hominis, Actinobacillus indolicus, Actinobacillus spp., Actinobacillus minor, Actinobacillus muris, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus scotiae, Actinobacillus seminis, Actinobacillus succinogenes, Actinobacillus ureae, Actinomyces bovis, Actinomyces bowdenii, Actinomyces canis, Actinomyces cardijfensis, Actinomyces catuli, Actinomyces coleocanis, Actinomyces dentalis, Actinomyces denticolens, Actinomyces europaeus, Actinomyces funkei, Actinomyces georgiae, Actinomyces graevenitzii, Actinomyces hongkongensis, Actinomyces hordeovulneris, Actinomyces howellii, Actinomyces hyovaginalis, Actinomyces marimammalium, Actinomyces massiliensis, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces nasicola, Actinomyces odontolyticus, Actinomyces radicidentis, Actinomyces radingae, Actinomyces ruminicola, Actinomyces slackii, Actinomyces suimastitidis, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces vaccimaxillae, Actinomyces viscosus, Aeromonas spp., Bacillus acidiceler, Bacillus spp., Bacillus acidicola, Bacillus aeolius, Bacillus aerius, Bacillus spp., Bacillus agar adhaer ens, Bacillus akibai, Bacillus alcalophilus, Bacillus algicola, Bacillus alkalidiazatrophicus, Bacillus alkalinitrilicus, Bacillus alkalitelluris, Bacillus alveayuensis, Bacillus aquimaris, Bacillus arseniciselenatis, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantiacus, Bacillus azatoformans, Bacillus badius, Bacillus barbaricus, Bacillus bataviensis, Bacillus benzaevorans, Bacillus bogoriensis, Bacillus butanolivorans, Bacillus carboniphilus, Bacillus cecembensis, Bacillus cellulosilyticus, Bacillus circulans, Bacillus clarkii, Bacillus clausii, Bacillus coagulans, Bacillus coahuilensis, Bacillus cohnii, Bacillus cytotoxicus, Bacillus decolorationis, Bacillus drentensis, Bacillus edaphicus, Bacillus endophyticus, Bacillus farraginis, Bacillus fastidiosus, Bacillus firmus, Bacillus flexus, Bacillus foraminis, Bacillus fordii, Bacillus fords, Bacillus fumarioli, Bacillus funiculus, Bacillus galactosidilyticus, Bacillus gelatini, Bacillus ginseng, Bacillus ginsengihumi, Bacillus halmapalus, Bacillus halodurans, Bacillus hemicellulosilyticus, Bacillus herbersteinensis, Bacillus horikoshii, Bacillus horti, Bacillus humi, Bacillus hwajinpoensis, Bacillus idriensis, Bacillus infantis, Bacillus infernus, Bacillus isabeliae, Bacillus koreensis, Bacillus korlensis, Bacillus kribbensis, Bacillus krulwichiae, Bacillus lentus, Bacillus licheniformis, Bacillus litoralis, Bacillus luciferensis, Bacillus macauensis, Bacillus macyae, Bacillus mannanilyticus, Bacillus marisflavi, Bacillus massiliensis, Bacillus methanolicus, Bacillus mucilaginosus, Bacillus murimartini, Bacillus nealsonii, Bacillus niabensis, Bacillus niacini, Bacillus novalis, Bacillus odyssey, Bacillus okhensis, Bacillus okuhidensis, Bacillus oleronius, Bacillus oshimensis, Bacillus panaciterrae, Bacillus plakortidis, Bacillus pocheonensis, Bacillus polygoni, Bacillus pseudalcaliphilus, Bacillus pseudofirmus, Bacillus psychrosaccharolyticus, Bacillus ruris, Bacillus schlegelii, Bacillus selenatarsenatis, Bacillus seohaeanensis, Bacillus shackletonii, Bacillus siralis, Bacillus smithii, Bacillus soli, Bacillus solisalsi, Bacillus sonorensis, Bacillus sp. I. MG 20238, Bacillus sporothermodurans, Bacillus taeanensis, Bacillus thermoamylovorans, Bacillus thermocloaceae, Bacillus thioparans, Bacillus vedderi, Bacillus vietnamensis, Bacillus vireti, Bacillus wakoensis, Bacteroides acidifaciens, Bacteroides barnesiae, Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides cellulosolvens, Bacteroides coprocola, Bacteroides coprophilus, Bacteroides coprosuis, Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides gallinarum, Bacteroides graminisolvens, Bacteroides helcogenes, Bacteroides helcogenes, Bacteroides heparinolyticus, Bacteroides intestinalis, Bacteroides massiliensis, Bacteroides nordii, Bacteroides ovatus, Bacteroides plebeius, Bacteroides propionicifaciens, Bacteroides pyogenes, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides xylanisolvens, Brucella spp., Burkholderia spp., Campylobacter spp., Cardiobacterium valvarum, Citrobacter braakii, Citrobacter freundii, Citrobacter gillenii, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Clostridium spp., Clostridium difficile, Clostridium perfringens, Corynebacterium spp., Corynebacterium accolens, Corynebacterium afermentans, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium appendicis, Corynebacterium aquilae, Corynebacterium atypicum, Corynebacterium aurimucosum, Corynebacterium auris, Corynebacterium auriscanis, Corynebacterium bovis, Corynebacterium callunae, Corynebacterium camporealensis, Corynebacterium capitovis, Corynebacterium casei, Corynebacterium caspium, Corynebacterium ciconiae, Corynebacterium confusum, Corynebacterium coyleae, Corynebacterium cystitidis, Corynebacterium diphtheriae, Corynebacterium durum, Corynebacterium efficiens, Corynebacterium falsenii, Corynebacterium felinum, Corynebacterium flavescens, Corynebacterium freiburgense, Corynebacterium freneyi, Corynebacterium glaucum, Corynebacterium glutamicum, Corynebacterium glutamicum, Corynebacterium halotolerans, Corynebacterium hansenii, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium kutscheri, Corynebacterium lipophiloflavum, Corynebacterium lubricantis, Corynebacterium macginleyi, Corynebacterium massiliense, Corynebacterium mastitidis, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium mycetoides, Corynebacterium phocae, Corynebacterium pilosum, Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium resistens, Corynebacterium riegelii, Corynebacterium simulans, Corynebacterium singulare, Corynebacterium sphenisci, Corynebacterium spheniscorum, Corynebacterium sputi, Corynebacterium striatum, Corynebacterium suicordis, Corynebacterium sundsvallense, Corynebacterium terpenotabidum, Corynebacterium testudinoris, Corynebacterium thomssenii, Corynebacterium timonense, Corynebacterium tuberculostearicum, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium ulceribovis, Corynebacterium urealyticum, Corynebacterium ureicelerivorans, Corynebacterium variabile, Corynebacterium xerosis, Dermatophilus congolensis, Edwardsiella spp., Enterobacter aerogenes, Enterobacter spp., Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Eubacterium acidaminophilum, Eubacterium angustum, Eubacterium brachy, Eubacterium budayi, Eubacterium cellulosolvens, Eubacterium desmolans, Eubacterium eligens, Eubacterium infirmum, Eubacterium minutum, Eubacterium multiforme, Eubacterium nitritogenes, Eubacterium nodatum, Eubacterium pyruvativorans, Eubacterium rectale, Eubacterium ruminantium, Eubacterium saphenum, Eubacterium sulci, Flavobacterium anhuiense, Flavobacterium antarcticum, Flavobacterium aquatile, Flavobacterium aquidurense, Flavobacterium ceti, Flavobacterium cheniae, Flavobacterium chungangense, Flavobacterium columnare, Flavobacterium croceum, Flavobacterium cucumis, Flavobacterium daejeonense, Flavobacterium defluvii, Flavobacterium degerlachei, Flavobacterium denitrificans, Flavobacterium filum, Flavobacterium frigidarium, Flavobacterium frigidimaris, Flavobacterium frigoris, Flavobacterium fryxellicola, Flavobacterium gelidilacus, Flavobacterium glaciei, Flavobacterium granuli, Flavobacterium hercynium, Flavobacterium hibernum, Flavobacterium hydatis, Flavobacterium indicum, Flavobacterium indicum, Flavobacterium johnsoniae, Flavobacterium limicola, Flavobacterium lindanitolerans, Flavobacterium micromati, Flavobacterium omnivorum, Flavobacterium psychrolimnae, Flavobacterium psychrophilum, Flavobacterium resistens, Flavobacterium saccharophilum, Flavobacterium saliperosum, Flavobacterium sasangense, Flavobacterium segetis, Flavobacterium soli, Flavobacterium sp. strain A103, Flavobacterium sp. strain ICOOl, Flavobacterium succinicans, Flavobacterium suncheonense, Flavobacterium terrae, Flavobacterium terrigena, Flavobacterium w eaver ense, Flavobacterium xanthum, Flavobacterium xinjiangense, Haemophilus spp., Haemophilus ducreyi, Haemophilus felis, Haemophilus haemoglobinophilus, Haemophilus influenzae, Haemophilus paracuniculus, Haemophilus parainfluenzae, Haemophilus paraphrohaemolyticus, Haemophilus parasuis, Haemophilus pittmaniae, Haemophilus somnus, Klebsiella spp., Lactobacillus acetotolerans, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus spp., Lactobacillus algidus, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus apodemi, Lactobacillus aviarius, Lactobacillus bobalius, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus catenaformis, Lactobacillus ceti, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus equi, Lactobacillus fabifermentans, Lactobacillus farraginis, Lactobacillus fornicalis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus ghanensis, Lactobacillus graminis, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus hordei, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefiri, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillus nagelii, Lactobacillus nantensis, Lactobacillus nodensis, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri, Lactobacillus perolens, Lactobacillus rapi, Lactobacillus rennini, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus satsumensis, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus spicheri, Lactobacillus suebicus, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini, Lactobacillus vitulinus, Listeria monocytogenes, Micrococcus antarcticus, Micrococcus endophyticus, Micrococcus flavus, Micrococcus spp., Micrococcus lylae, Mobiluncus curtisii, Mobiluncus curtisii subsp. curtisii, Mobiluncus curtisii subsp. holmesii, Mobiluncus mulieris, Moraxella boevrei, Moraxella bovis, Moraxella bovoculi, Moraxella canis, Moraxella caprae, Moraxella catarrhalis, Moraxella caviae, Moraxella cuniculi, Moraxella equi, Moraxella lacunata, Moraxella ovis, Moraxella pluranimalium, Morganella morganii, Morganella psychrotolerans, Mycobacterium massiliense, Mycobacterium sp, Mycobacterium tuberculosis, Neisseria animalis, Neisseria animaloris, Neisseria bacilliformis, Neisseria canis, Neisseria dentiae, Neisseria elongata subsp. elongate, Neisseria flavescens, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria meningitides, Neisseria polysaccharea, Neisseria subflava, Neisseria weaveri, Neisseria zaodegmatis, Nocardia abscessus, Nocardia acidivorans, Nocardia africana, Nocardia alba, Nocardia altamirensis, Nocardia amamiensis, Nocardia anaemiae, Nocardia aobensis, Nocardia araoensis, Nocardia arthritidis, Nocardia asiatica, Nocardia asteroides, Nocardia beijingensis, Nocardia blacklockiae, Nocardia brasiliensis, Nocardia brevicatena, Nocardia caishijiensis, Nocardia carnea, Nocardia cerradoensis, Nocardia concave, Nocardia coubleae, Nocardia crassostreae, Nocardia cyriacigeorgica, Nocardia elegans, Nocardia exalbida, Nocardia flavorosea, Nocardia fluminea, Nocardia gamkensis, Nocardia harenae, Nocardia higoensis, Nocardia ignorata, Nocardia inohanensis, Nocardia jejuensis, Nocardia jiangxiensis, Nocardia jinanensis, Nocardia lijiangensis, Nocardia miyunensis, Nocardia neocaledoniensis, Nocardia niigatensis, Nocardia ninae, Nocardia nova, Nocardia otitidiscaviarum, Nocardia paucivorans, Nocardia pneumoniae, Nocardia pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia puris, Nocardia salmonicida, Nocardia seriolae, Nocardia shimofusensis, Nocardia speluncae, Nocardia takedensis, Nocardia tenerifensis, Nocardia terpenica, Nocardia thailandica, Nocardia transvalensis, Nocardia uniformis, Nocardia vaccinii, Nocardia vermiculata, Nocardia vinacea, Nocardia wallacei, Nocardia xishanensis, Nocardia yamanashiensis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Porphyromonas bennonis, Porphyromonas cangingivalis, Porphyromonas cansulci, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gulae, Porphyromonas levii, Porphyromonas somerae, Porphyromonas uenonis, Prevotella albensis, Prevotella amnii, Prevotella baroniae, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella corporis, Prevotella denticola, Prevotella disiens, Prevotella enoeca, Prevotella falsenii, Prevotella histicola, Prevotella intermedia, Prevotella loescheii, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella salivae, Prevotella shahii, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella veroralis, Propionibacterium acidipropionici, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium cyclohexanicum, Propionibacterium freudenreichii, Propionibacterium freudenreichii subsp. shermanii, Propionibacterium granulosum, Propionibacterium jensenii, Propionibacterium microaerophilum, Propionibacterium propionicum, Propionibacterium thoenii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia heimbachae, Providencia rettgeri, Providencia rustigianii, Providencia vermicola, Pseudomonas aeruginosa, Rahnella aquatilis,Rhodococcus aetherivorans, Rhodococcus coprophilus, Rhodococcus corynebacterioides, Rhodococcus erythropolis, Rhodococcus fascians, Rhodococcus globerulus, Rhodococcus gordoniae, Rhodococcus imtechensis, Rhodococcus jostii, Rhodococcus koreensis, Rhodococcus kroppenstedtii, Rhodococcus kunmingensis, Rhodococcus kyotonensis, Rhodococcus maanshanensis, Rhodococcus marinonascens, Rhodococcus opacus, Rhodococcus percolatus, Rhodococcus phenolicus, Rhodococcus pyridinivorans, Rhodococcus qingshengii, Rhodococcus rhodnii, Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus triatomae, Rhodococcus tukisamuensis, Rhodococcus wratislaviensis, Rhodococcus yunnanensis, Rhodococcus zapfii, Salmonella bongori, Salmonella spp., Serratia spp., Shigella spp., Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus spp. Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, Streptococcus caballi, Streptococcus canis, Streptococcus castoreus, Streptococcus criceti, Streptococcus cristatus, Streptococcus dentirousetti, Streptococcus devriesei, Streptococcus didelphis, Streptococcus downei, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus entericus, Streptococcus equi subsp. equi, Streptococcus equi subsp. ruminatorum, Streptococcus equi subsp. zaoepidemicus, Streptococcus ferus, Streptococcus gallinaceus, Streptococcus gallolyticus subsp. gallolyticus, Streptococcus gordonii, Streptococcus halichoeri, Streptococcus henryi, Streptococcus hyointestinalis, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedins, Streptococcus luteciae, Streptococcus macacae, Streptococcus macedonicus, Streptococcus marimammalium, Streptococcus massiliensis, Streptococcus merionis, Streptococcus minor, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus orisratti, Streptococcus orisuis, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus plurextorum, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus ratti, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus sp. strain SHV515, Streptococcus suis, Streptococcus thermophilus, Streptococcus thoraltensis, Streptococcus uberis, Streptococcus urinalis, Streptococcus viridans, Streptomyces achromogenes subsp. rubradiris, Streptomyces acidiscabies, Streptomyces aculeolatus, Streptomyces alanosinicus, Streptomyces albiaxialis, Streptomyces albiflaviniger, Streptomyces albofaciens, Streptomyces alboflavus, Streptomyces alboniger, Streptomyces albospinus, Streptomyces albosporeus subsp. labilomyceticus, Streptomyces albovinaceus, Streptomyces alni, Streptomyces ambofaciens, Streptomyces anandii, Streptomyces antibioticus, Streptomyces ardus, Streptomyces armeniacus, Streptomyces asiaticus, Streptomyces atroaurantiacus, Streptomyces atrovirens, Streptomyces aurantiogriseus, Streptomyces auratus, Streptomyces aureocirculatus, Streptomyces aureoverticillatus, Streptomyces aureus, Streptomyces avermitilis, Streptomyces axinellae, Streptomyces azureus, Streptomyces bacillaris, Streptomyces beijiangensis, Streptomyces bikiniensis, Streptomyces bingchenggensis, Streptomyces blastmyceticus, Streptomyces bluensis, Streptomyces bottropensis, Streptomyces brasiliensis, Streptomyces cacaoi subsp. asoensis, Streptomyces cacaoi subsp. cacaoi, Streptomyces caelestis, Streptomyces caeruleus, Streptomyces candidus, Streptomyces capoamus, Streptomyces carpaticus, Streptomyces carpinensis, Streptomyces catenulae, Streptomyces chartreusis, Streptomyces cheonanensis, Streptomyces chromofuscus, Streptomyces cinereoruber subsp. cinereoruber, Streptomyces cinereoruber subsp. fructofermentans, Streptomyces cinereospinus, Streptomyces cinereus, Streptomyces cinerochromogenes, Streptomyces clavuligerus, Streptomyces coelicoflavus, Streptomyces coeruleofuscus, Streptomyces coeruleoprunus, Streptomyces coeruleorubidus, Streptomyces crystallinus, Streptomyces cyaneus, Streptomyces cyanoalbus, Streptomyces deccanensis, Streptomyces diastatochromogenes, Streptomyces djakartensis, Streptomyces drozdowiczii, Streptomyces durmitorensis, Streptomyces ehimensis, Streptomyces emeiensis, Streptomyces eurocidicus, Streptomyces eurythermus, Streptomyces ferralitis, Streptomyces filamentosus, Streptomyces fimbriatus, Streptomyces fimicarius, Streptomyces flaveolus, Streptomyces flaveus, Streptomyces flavofimgini, Streptomyces flavogriseus, Streptomyces flavovirens, Streptomyces flocculus, Streptomyces fradiae, Streptomyces fragilis, Streptomyces fumanus, Streptomyces fumigatiscleroticus, Streptomyces geldanamycininus, Streptomyces gibsonii, Streptomyces glaucescens, Streptomyces glauciniger, Streptomyces glaucosporus, Streptomyces glaucus, Streptomyces glomeratus, Streptomyces griseoluteus, Streptomyces griseoruber, Streptomyces griseosporeus, Streptomyces guanduensis, Streptomyces gulbar gensis, Streptomyces hainanensis, Streptomyces hawaiiensis, Streptomyces hebeiensis, Streptomyces heliomycini, Streptomyces himastatinicus, Streptomyces hiroshimensis, Streptomyces hirsutus, Streptomyces humidus, Streptomyces hypolithicus, Streptomyces indiaensis, Streptomyces ipomoeae, Streptomyces jietaisiensis, Streptomyces kanamyceticus, Streptomyces katrae, Streptomyces koyangensis, Streptomyces kunmingensis, Streptomyces lanatus, Streptomyces laurentii, Streptomyces lavenduligriseus, Streptomyces levis, Streptomyces lilacinus, Streptomyces lincolnensis, Streptomyces litmocidini, Streptomyces lomondensis, Streptomyces longispororuber, Streptomyces longisporus, Streptomyces lucensis, Streptomyces lunalinharesii, Streptomyces luridiscabiei, Streptomyces lusitanus, Streptomyces luteireticuli, Streptomyces luteogriseus, Streptomyces luteosporeus, Streptomyces macrosporus, Streptomyces malachitofuscus, Streptomyces malachitospinus, Streptomyces malaysiensis, Streptomyces mashuensis, Streptomyces massasporeus, Streptomyces mayteni, Streptomyces megasporus, Streptomyces mexicanus, Streptomyces minutiscleroticus, Streptomyces mirabilis, Streptomyces misakiensis, Streptomyces mobaraensis, Streptomyces monomycini, Streptomyces morookaensis, Streptomyces naganishii, Streptomyces nanshensis, Streptomyces neyagawaensis, Streptomyces nitrosporeus, Streptomyces niveiscabiei, Streptomyces nodosus, Streptomyces nogalater, Streptomyces novaecaesareae, Streptomyces odorifer, Streptomyces olivochromogenes, Streptomyces olivoverticillatus, Streptomyces orinoci, Streptomyces paradoxus, Streptomyces parvulus, Streptomyces paucisporeus, Streptomyces phaeochromogenes, Streptomyces phaeofaciens, Streptomyces phaeogriseichromatogenes, Streptomyces phaeoluteigriseus, Streptomyces phaeopurpureus, Streptomyces pharetrae, Streptomyces plumbiresistens, Streptomyces polyantibioticus, Streptomyces poonensis, Streptomyces prasinopilosus, Streptomyces prasinosporus, Streptomyces prunicolor, Streptomyces psammoticus, Streptomyces pseudovenezuelae, Streptomyces puniciscabiei, Streptomyces purpeofuscus, Streptomyces purpurascens, Streptomyces purpureus, Streptomyces radiopugnans, Streptomyces ratnulosus, Streptomyces rangoonensis, Streptomyces rectiviolaceus, Streptomyces regensis, Streptomyces reticuliscabiei, Streptomyces rishiriensis, Streptomyces roseofulvus, Streptomyces roseolilacinus, Streptomyces roseolus, Streptomyces roseoviridis, Streptomyces rubidus, Streptomyces rubiginosus, Streptomyces sampsonii, Streptomyces scabrisporus, Streptomyces sclerotialus, Streptomyces scopiformis, Streptomyces sedi, Streptomyces sioyaensis, Streptomyces sodiiphilus, Streptomyces sp. 40003, Streptomyces sp. SirexAA-E, Streptomyces sp. strain ISP 5133, Streptomyces sp. strain ISP 5310, Streptomyces sp. strain ISP 5499, Streptomyces specialis, Streptomyces speibonae, Streptomyces spinoverrucosus, Streptomyces spiralis, Streptomyces stelliscabiei, Streptomyces stramineus, Streptomyces sulfonofaciens, Streptomyces sulphureus, Streptomyces synnetnatofortnans, Streptomyces tauricus, Streptomyces termitum, Streptomyces thermoalcalitolerans, Streptomyces thermocarboxydovorans, Streptomyces thermocarboxydus, Streptomyces thermocoprophilus, Streptomyces thermodiastaticus, Streptomyces thermolineatus, Streptomyces thermospinosisporus, Streptomyces thermoviolaceus subsp. thermoviolaceus, Streptomyces thioluteus, Streptomyces tuirus, Streptomyces turgidiscabies, Streptomyces varsoviensis, Streptomyces vastus, Streptomyces vietnamensis, Streptomyces violaceorectus, Streptomyces viridiviolaceus, Streptomyces viridochromogenes, Streptomyces viridosporus, Streptomyces vitaminophilus, Streptomyces xiamenensis, Streptomyces yanglinensis, Streptomyces yatensis, Streptomyces yeochonensis, Streptomyces yerevanensis, Streptomyces yokosukanensis, Vibrio spp., Vibrio fluvialis, Vibrio metschnikovii, Vibrio parahaernolyticus, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia spp.
[0070] In any and all embodiments of the methods disclosed herein, the subject is an immunocompromised subject, a pediatric subject, a geriatric subject, or an adult subject.
Kits of the Present Technology
[0071] The present disclosure provides kits comprising a fungal beta-glucan (e.g., Saccharomyces cerevisiae beta-glucan), an alkalinization agent (e.g., borate buffer, an alkali- metal hydroxide, or an alkali earth-metal hydroxide as described herein) and instructions for using the same to treat or prevent infection-induced sepsis in a subject in need thereof, wherein the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l,3) backbone via p~( 1 ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol. In certain embodiments, the kits of the present technology comprise instructions for alkali-treating the fungal beta-glucan prior to immunizing the subject.
[0072] Additionally or alternatively, in some embodiments of the kits, the alkalinized fungal beta-glucan is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral or intranasal administration.
[0073] Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for preventing or treating infection-induced sepsis (e.g., polymicrobial infection) in a subject. The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, etc. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.
[0074] The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. In certain embodiments, the use of the reagents can be according to the methods of the present technology.
EXAMPLES
[0075] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.
Example 1: Materials and Experimental methods
[0076] A. Innate Immune Protection Studies Against Bacterial/Fungal Infection-induced Sepsis
[0077] Mice. For all experiments, female Swiss Webster mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc. Animals were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following I ACUC -approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
[0078] Microbial Strains and growth conditions. C. albicans strain DAY185, a prototrophic derivative of SC5314, was a gift from Aaron Mitchell (Carnegie Mellon University, Pittsburgh, PA). The C. dubliniensis wild-type strain (Wu284) was kindly provided by Gary Moran (Trinity College, Dublin, Ireland). Frozen stocks were maintained at -80°C and streaked onto yeast extract-peptone-dextrose (YPD) agar prior to use. A single colony was transferred to 10 ml of YPD broth, and the culture was shaken at 30°C for 12 to 18 h. The methicillin-resistant S. aureus strain NRS383 used in all experiments was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) data bank. Frozen stocks were maintained at -80°C and streaked onto Trypticase soy agar (TSA) prior to use. The E. coli strain 25922 (ATCC) used in all experiments, a gift from Jacob Bitoun (Tulane University School of Medicine, New Orleans, LA), was chosen based on previous publications documenting sepsis or peritonitis models (Cirioni et al., 2006; Chen et al., 2010; Ghiselli et al., 2011). The strain ATCC 25922 is a commonly used quality control strain, particularly in antibody sensitivity assays and was originally isolated from a human clinical sample collected in Seattle and WA (1946). It is of serotype 06 and biotype 1.
[0079] Frozen stocks were maintained at -80°C and streaked onto Trypticase soy agar (TSA) prior to use. For both bacterial pathogens, a single colony was transferred to 10 ml of Trypticase soy broth (TSB) or Luria Broth (LB) and shaken at 37°C overnight. On the following day, the overnight culture was diluted 1 : 100 in fresh TSB/LB and shaken at 37°C for 1.5-3 h until the culture reached the log phase of growth. Prior to inoculation, cultures of both organisms were washed 3 times by centrifugation in sterile PBS (pH 7.4), counted using a hemocytometer, and diluted in sterile PBS to prepare standardized inocula.
[0080] Immunizations. For all immunization experiments, groups (n = 4-10) of 6-week-old outbred Swiss Webster or inbred C57BL/6J were used.
[0081] Live C. dubliniensis (Cd). Mice were inoculated i.p. with 200 ul Cd (1.75 * 107/mouse) resuspended in sterile non-pyrogenic PBS 14 d prior to sepsis challenge.
[0082] Abiotic Fungal cell wall compounds. Mice were injected i.p. with various P-glucan preparations prior to challenge in sepsis models. Purified P-glucan from S. cerevisiae (Millipore, cat# 346210): Mice were injected with 1 dose of 1-4 mg untreated or modified P-glucan 7-14 days prior to sepsis challenge. To produce modified P-glucan, purified P-glucan was alkali treated by overnight incubation at room temperature in 0.1 M borate buffer (pH = 9.8). The solution was centrifuged, and pellet washed 3 times in sterile PBS, and resuspended in 0.2 N NaOH and incubated for 20 min at room temperature. The solution was centrifuged, and pellet washed 3 times with sterile DI H2O and resuspended in sterile non-pyrogenic PBS prior to injection.
[0083] Depleted Zymosan from S. cerevisiae (zymosan-D; Invivogen, cat# tlrl-zy d): Mice were injected with 1 (day -14) or 2 (day -14, day -7) doses of 1.2 mg zymosan-D dissolved in sterile non-pyrogenic PBS prior to sepsis challenge.
[0084] Whole glucan particulate (WGP dispersible; Invivogen, cat#tlrl-wgp): Mice were injected with 1 dose of 200pg WGP dissolved in sterile non-pyrogenic PBS 14 days prior to sepsis challenge. The concentrations of P-glucans (molecular and WGP dispersible) were based on studies by Moorlag et al. where in vivo trained innate immune responses were elicited in mice who had been immunized intraperitoneally with 1 mg P-glucan (Moorlag et al., 2020b).
Increased doses were tested to optimize protective effects. Highest efficacy against polymicrobial challenge was was observed using one dose of 4 mg P-glucan.
[0085] Murine models of sepsis. Polymicrobial IAI: Mice were injected i.p. with a lethal challenge of C. albicans (1.75 x 107/mouse) and A. coli (4.5 x 106/mouse) in a volume of 200 pl. In both aseptic and polymicrobial rechallenge models the mice were observed for morbidity using the modified M-CASS (scores consider a range from 0-3 when observing aspects such as fur ruffling, activity level, posture/hunching, behavior, respiration quality/rate and squinting or orbital tightening) and mortality up to 10 days after challenge (Mai et al., 2018). Mice who reached clinical endpoints prior to study endpoint were humanely euthanized following IACUC- approved euthanasia procedures. To assess microbial burdens, spleens (homogenized in 500 pl sterile PBS) and 1 ml peritoneal lavage fluid were collected from each mouse at clinical or study endpoint. CFUs were enumerated by plating serial diluations of spleen homogenates or peritoneal lavage samples onto YPD agar containing 40 pg/ml gentamycin and 2 pg/ml vancomycin for fungal colonies and TSA agar containing 40 pg/ml gentamycin and 2.5 pg/ml amphotericin B for bacterial colonies. Plates were incubated overnight at 37°C. CFU counts are expressed as the number of CFU/ml of peritoneal lavage fluid and CFU/g of spleen homogenates. Based on dilution volumes, the limit of detection for peritoneal lavage fluid and spleens are 20 and 50 CFU, respectively. [0086] Zymosan: Mice were injected i.p. with a lethal challenge of 700-1000 mg/kg of zymosan obtained from S. cerevisiae (Sigma Aldrich) and resuspended in sterile NaCl.
[0087] LPS: Mice were injected i.p. with a lethal challenge of 300 pg (10 mg/kg) of LPS obtained from E. coll Ol l i :B4 (Sigma Aldrich) and resuspended in sterile NaCl.
[0088] Cell Depletion. For macrophage depletion, liposome-encapsulated clodronate and liposome vehicle (1 mg/mouse; Encapsula NanoSciences) were injected i.p. in 200 pl 1 day prior to sepsis challenge. Clodronate (dichloromethylene-bisphosphonate) is encapsulated in the aqueous compartments of liposomes which have been filtered for size to remove larger particles that might be toxic to animals. The liposomal solution is injected intraperitoneally into the mice where phagocytic cells recognize the liposomes as foreign particles and phagocytose them. When internalized, the liposomes release clodronate into the cytosol, resulting in cell death. Liposomes without clodronate exhibit no cellular toxicity.
[0089] For granulocyte depletion, mice were injected i.p. with either 200 pg rat anti-mouse Gr-1+ (Ly6G/Ly6C) or rat IgG2A isotype control antibodies (Bio-X-Cell) in 200 pl sterile non- pyrogenic PBS to systemically deplete PMNLs 48 h prior to and 2 h after challenge. Injections were given every 2 days for the duration of the study. Depletion was confirmed by flow cytometry.
[0090] Sepsis Scoring. Mice are monitored for survival in three checks per day following challenge for 10 days. Daily behavioral scoring is performed using a modified sepsis scoring criteria including fur aspect, activity level, posture, breathing quality and grimace signs to quantify morbidity and predict mortality in mice (Mai et al., 2018). Onset of sepsis is rapid in unvaccinated mice; therefore, scoring in these groups is monitored for all mice through study or clinical endpoint but only reported up to the time that the majority of animals in the group have been euthanized to eliminate presentation of results that are not representative of the group as a whole.
[0091] Statistics. Survival curves were compared using the log rank (Mantel-Cox) test. Significant differences were defined at <0.05. Sepsis scores were compared using ANOVA followed by post hoc Student’s t-test. For microbial burden assessments, calculated CFU counts were log transformed for the purpose of normalization and analyzed using a one-way ANOVA with Tukey’s test for multiple comparisons. These statistical analyses were performed using Prism software (Graph Pad Prism 9). [0092] B. Pattern Recognition Receptor & Cytokine Analysis
[0093] Mice. For all experiments, female Swiss Webster mice, 5 to 7 weeks of age, or female C57BL/6 mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc or Jackson Laboratories, respectively. All PRR knockout mice were developed on a C57BL/6 background and obtained from Jackson Laboratories or collaborating laboratories (i.e., Dectin- 1" A, B6.129S6-Clec7atmlGdb/J; MyD88'/_, B6.129P2(SJL)-MyD88tol 1Def7J; CARDO'7', B6.129- Card9tolxlin/J; and IL-10'7', B6.129P2-I110tmlCgn/J). Animals were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following JACUC-approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
[0094] Microbial Strains and growth conditions. C. albicans strain DAY185, a prototrophic derivative of SC5314, was a gift from Aaron Mitchell (Carnegie Mellon University, Pittsburgh, PA). The C. dubliniensis wild-type strain (Wu284) was kindly provided by Gary Moran (Trinity College, Dublin, Ireland). Frozen stocks were maintained at -80°C and streaked onto yeast extract-peptone-dextrose (YPD) agar prior to use. A single colony was transferred to 10 ml of YPD broth, and the culture was shaken at 30°C for 12 to 18 h.
[0095] The methicillin-resistant S. aureus strain NRS383 used in all experiments was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) data bank. Frozen stocks were maintained at -80°C and streaked onto Trypticase soy agar (TSA) prior to use. For the bacterial pathogen, a single colony was transferred to 10 ml of Trypticase soy broth (TSB) and shaken at 37°C overnight. On the following day, the overnight culture was diluted 1 : 100 in fresh TSB and shaken at 37°C for 2-3 h until the culture reached the log phase of growth. Prior to inoculation, cultures were washed 3 times by centrifugation in sterile PBS (pH 7.4), counted using a hemocytometer, and diluted in sterile PBS to prepare standardized inocula.
[0096] Abiotic f-glucan Methods of Preparation and Quantification. To produce modified
P-glucan, purified P-glucan from S. cerevisiae was alkali treated by overnight incubation at room temperature in 0.1 M borate buffer (pH = 9.8). The solution was centrifuged, and the pellet was washed 3 times in sterile PBS, resuspended in 0.2 N NaOH, and incubated for 20 min at room temperature. The solution was then centrifuged, and the pellet was washed 3 times with sterile DI H2O and resuspended in sterile non-pyrogenic PBS prior to injection. [0097] Immunizations. For all immunization experiments, groups (n = 3-10) of 6-week-old outbred Swiss Webster or inbred C57BL/6J were used.
[0098] Live C. dubliniensis (Cd).' Mice were inoculated i.p. with 200 pl Cd (1.75 * 107/mouse) resuspended in sterile non-pyrogenic PBS 14 d prior to sepsis challenge.
[0099] Abiotic fungal cell wall compounds: Mice were injected i.p. with 1 dose (day -7) of 4 mg modified P-glucan preparations (prepared as described above) or 2 doses (day -14, day -7) of 1.2 mg d-zymosan (depleted zymosan from S. cerevisiae Invivogen, cat# tlrl-zyd) dissolved in sterile non-pyrogenic PBS prior to sepsis challenge.
[00100] Murine model of sepsis. Polymicrobial IAI: Mice were injected i.p. with a lethal challenge of C. albicans (1.75 x 107/mouse) and S. aureus (8 x 107/mouse) in a volume of 200 pl. In both aseptic and polymicrobial rechallenge models the mice were observed for morbidity using the modified M-CASS (scores consider a range from 0-3 when observing aspects such as fur ruffling, activity level, posture/hunching, behavior, respiration quality/rate and squinting or orbital tightening) and mortality up to 10 days after challenge (Mai et al., 2018). Mice who reached clinical endpoints prior to study endpoint were humanely euthanized following IACUC- approved euthanasia procedures. To assess microbial burdens, spleens (homogenized in 500 pl sterile PBS) and 1 ml peritoneal lavage fluid were collected from each mouse at clinical or study endpoint. CFUs were enumerated by plating serial dilutions of spleen homogenates or peritoneal lavage samples onto YPD agar containing 40 pg/ml gentamycin and 2 pg/ml vancomycin for fungal colonies and TSA agar containing 40 pg/ml gentamycin and 2.5 pg/ml amphotericin B for bacterial colonies. Plates were incubated overnight at 37°C. CFU counts are expressed as the number of CFU/ml of peritoneal lavage fluid and CFU/g of spleen homogenates. Based on dilution volumes, the limit of detection for peritoneal lavage fluid and spleens are 20 and 50 CFU, respectively.
[00101] Sepsis Scoring. Mice are monitored for survival for 10 days post-lethal challenge in three checks daily. Daily behavioral scoring is performed using a modified sepsis scoring criteria including fur aspect, activity level, posture, breathing quality and grimace signs to quantify morbidity and predict mortality in mice (Mai et al., 2018). Onset of sepsis is rapid in unimmunized mice; therefore, scoring in these groups is monitored for all mice through study or clinical endpoint but only reported up to the time that the majority of animals in the group have been euthanized to eliminate presentation of results that are not representative of the group as a whole. [00102] Cell Culture. HEK-Blue PRR reporter cell lines (hDectin-1 A, cat#hkb-hdectla; mDectin-2, cat#hkb-mdect2; mTLR-2, cat#hkb-mtlr2, Invivogen; hTLR-4, cat#hkb-htlr4, Invivogen) were maintained in cell culture medium at 37°C, 5% CO2. HEK-BLUE-mTLR2, HEK-BLUE-mDectin-2 and HEK-BLUE-hDectin-1 A cells were cultured in DMEM with 10% FBS, 100 pg/mL penicillin-streptomycin, 100 pg/mL normocin, 2mM L-glutamine and supplemented with IX HEK-BLUE Selection. HEK-BLUE-mDectin-2 and HEK-BLUE- hDectin-1 A cells were maintained with growth media supplemented with Puromycin with or without Blasticidin as per manufacturer instructions. HEK-BLUE-hTLR4 cells were maintained with growth media supplemented with Blasticidin as per manufacturer instructions.
[00103] Stimulation of Reporter Cells. Cells were plated at the density of 50,000 cells/well in flat bottom 96-well plates in a 180pL volume and 2-3 dilutions starting at 10-100 pg/mL of the respective vaccines and PRR ligands were prepared and added in a 20pL volume. Zymosan (10 or 100 pg/mL) or LPS (10 ng/mL) served as positive controls and PBS as a negative control for each experiment. Cells were incubated for 22 h at 37°C, 5% CO2, then quantified via spectrophotometer at 620 nm.
[00104] Milliplex Mouse 32-Plex. Mouse serum cytokine concentrations were determined using a multiplex immunoassay (cat# MCYTMAG-70K-PX32, Millipore) and analyzed using a BioPlex 200 (Bio-Rad, United States). Mouse cytokines and chemokines measured include: Eotaxin, G-CSF, GM-CSF, IFN-y, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-la, MIP-ip, MIP-2, RANTES, TNF, and VEGF. All protocols were performed according to manufacturer instructions.
[00105] Statistics. Survival curves were compared using the log rank (Mantel-Cox) test. Significant differences were defined at P<0.05. Sepsis scores and serum cytokine levels were compared using ANOVA followed by post hoc Student’s t-test. For microbial burden assessments, calculated CFU counts were log transformed for the purpose of normalization and analyzed using a one-way ANOVA with Tukey’s test for multiple comparisons. PRR signaling differences were compared using one-way ANOVA with all values compared to the PBS negative control. These statistical analyses were performed using Prism software (Graph Pad Prism 9).
[00106] C. Innate Immune Protection Studies Against Viral Infection-induced Sepsis [00107] Mice. For all experiments, female Swiss Webster mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories, Inc (Wilmington, MA). Animals were housed and handled according to institutionally recommended guidelines. Mice that reached clinical endpoints prior to study endpoint were humanely euthanized following lACUC-approved euthanasia procedures. All experiments involving animals were approved by the Tulane Institutional Animal Care and Use Committee.
[00108] Microbial Strains and Growth Conditions. C. dubliniensis wild-type strain (Wu284) was kindly provided by Gary Moran (Trinity College, Dublin, Ireland). Frozen stocks were maintained at -80°C and streaked onto yeast extract-peptone-dextrose (YPD) agar prior to use. A single colony was transferred to 10 ml of YPD broth, and the culture was shaken at 30°C for 12 to 18 h. Prior to inoculation, cultures were washed 3 times by centrifugation in sterile PBS (pH 7.4), counted using a hemocytometer, and diluted in sterile PBS to prepare standardized inocula.
[00109] H1N1. A/PR8/1934 (H1N1 PR8) is an H1N1 strain of influenza that was isolated from Puerto Rico and has been mouse adapted. It recapitulates the severe acute respiratory distress (ARDS) and diffuse alveolar damage (DAD) that is observed in humans during pandemic outbreaks. The virus was propagated in embryonated chicken eggs and was kindly provided by Derek Pociask (Tulane University, New Orleans, LA).
[00110] Immunizations. For all immunization experiments, groups (n = 4-10) of 6-week-old outbred Swiss Webster or inbred C57BL/6J were used. Mice were inoculated i.p. with 200 ul Cd (1.75 x 107/mouse) resuspended in sterile non-pyrogenic PBS 14 d prior to sepsis challenge.
[00111] Murine Model of Respiratory Infection.
[00112] Mice were anesthetized with isoflurane and oropharyngeally aspirated a lethal challenge of 15 PFU of H1N1 influenza and resuspended in sterile PBS. Influenza had been previously quantified by plaque assay in MDCK cells.
[00113] Cells were seeded on 24-well tissue culture treated plates (Greiner bio-one) with 3* 105 cells/0.5 ml/well. 24h later, cells were washed twice with sterile PBS then inoculated with influenza virus thawed and tenfold serially diluted in sterile PBS. The inoculum was adsorbed at RT for Ih with agitation every 10 min to maintain even plaque distribution. The cells were washed with sterile PBS and the overlay medium was added [2x TMEM (Gibco) containing 2x MEM, non-essential amino acids (Gibco), 4 mM 1-glutamine (Lonza), 0.2% bovine serum albumin (BSA, A0336 Sigma-Aldrich), 2 mM HEPES (BioWhittaker), 2x antibiotic-antimycotic solution (Gibco) with equivalent volume of 37 °C tempered 1.8% agarose (AGTC Bioproducts, gelling point of 1.5% agarose <27 °C) heat-dissolved in sterile PBS], Plates were incubated upside-down at 37°C, 5% CO2 for 72 h then fixed with 20% trichloroacetic acid (TCA). After 90 minutes, the overlay medium (with TCA) was removed, and cells were washed with PBS and permeabilized 10 min with absolute MeOH. Cells were washed 3x then stained with 1% crystal violet in 20% ethanol solution to visualize plaques. Wells with plaque counts >10 and <200 were used to calculate virus titer as plaque forming units per milliliter.
[00114] Cell Depletion. For macrophage depletion, liposome-encapsulated clodronate and liposome vehicle (1 mg/mouse; Encapsula NanoSciences) were injected i.p. in 200 pl 1 day prior to challenge or administered intranasally in 50pL 48h prior to and post-lethal challenge.
Clodronate (dichloromethylene-bisphosphonate) is encapsulated in the aqueous compartments of liposomes which have been filtered for size to remove larger particles that might be toxic to animals. The liposomal solution is administered to the mice where phagocytic cells recognize the liposomes as foreign particles and phagocytose them. When internalized, the liposomes release clodronate into the cytosol, resulting in cell death. Liposomes without clodronate (the control group) exhibit no cellular toxicity.
[00115] For granulocyte depletion, mice were injected i.p. with either 200 pg rat anti-mouse Gr-1+ (Ly6G/Ly6C) or rat IgG2A isotype control antibodies (Bio-X-Cell) in 200 pl sterile non- pyrogenic PBS to systemically deplete PMNLs 48 h prior to and 2 h after challenge. Injections were given every 2 days for the duration of the study. Depletion was previously confirmed in our laboratory by flow cytometry.
[00116] Sepsis Scoring/Weight Monitoring. Mice are monitored for survival in three checks per day for 15 days following challenge. Daily behavioral scoring is performed using a modified sepsis scoring criteria including fur aspect, activity level, posture, breathing quality and grimace signs to quantify morbidity and predict mortality in mice (Mai et al., 2018). Mice were weighed daily with values assessed as percentage of starting weight. Mice reached clinical endpoint at or greater than 30% loss of original weights.
[00117] Blood and Lung Harvest and Processing. Upon euthanization, blood was collected via direct cardiac puncture. This also served as a secondary method of euthanasia per IACUC protocols. Blood was incubated for 2h at 4°C then centrifuged at 13,000 RPM for 90 seconds to separate serum. Serum was stored in aliquots at -80°C prior to use. [00118] Lungs were washed with PBS using intratracheal instillation to remove red blood cells. One lung (2-3 lobes) was digested then processed to isolate immune cells (see below). The second lung (2-3 lobes) was placed in trizol and homogenized prior to RNA isolation. Homogenized lungs were stored at -80°C prior to RNA extraction.
[00119] RNA extraction and quantitation methods. Isopropanol was added into the homogenized tissue and trizol mixture (1 :2 ratio) and incubated for 10 minutes at 4°C. Tissues were spun at 12,000 x g for 10 minutes at 4°C then supernatant was removed via pipette and discarded. RNA pellet was resuspended in 1 mL 75% EtOH, vortexed and spun for 5 minutes at 7500 x g at 4°C. Supernatant was discarded and pellet was dried in a biosafety cabinet for 5-10 minutes. Pellet was resuspended in 50 pL of RNase-free water with 0.1 mM EDTA and incubated at 60°C for 15 minutes. RNA yield was measured via nanodrop via A260/A280 ratio.
[00120] RNA was reverse transcribed into DNA using High-capacity cDNA reverse transcription kit (cat# LT-02241, Applied Biosystems). To amplify DNA, 10 pL cDNA was added into 10 pL TaqMan master mix in a PCR reaction plate then run in an MJ Mini personal Thermal Cycler (Bio-Rad) for 40 cycles. Viral RNA quantification was calculated according to amplification plots. Threshold cycle (Ct) numbers were used to compare values as ACt and relative RNA expression.
[00121] Lung Cell Isolation. Washed lung tissue was cut into small pieces with surgical scissors then enzymatically digested for 45 min in 1 mL of RPMI with fetal bovine serum, heparin, liberase, EDTA, and DNASE 1 in a 12 well plate at 37°C with shaking. After enzymatic digestion, the contents of each well were pushed through a 70pm filter and washed with cold PBS with 10% FBS to stop the enzymatic process. Cell suspension was spun at 500x g for 5 minutes. Red blood cells were lysed using 1 mL of ACK (ammonium-chloride-potassium) lysing buffer for 5 min on ice and then washed with PBS twice. Cell suspension was resuspended in 2% paraformaldehyde and incubated on ice for 10 minutes to fix the cells.
[00122] Staining and Flow Cytometry Methods. After fixing cells with PF A, cells are washed twice with PBS, and spun at 500 x g. Tubes were decanted to remove supernatant then cells were counted, and 2xl06 cells per sample were stained with Live/Dead viability dye (cat#564406; BD Bioscience) according to the manufacturer’s instructions. Cells were then incubated in a blocking solution containing 0.5% BSA with CD16/CD32 to block Fc receptor non-specific binding (eBiosciences, San Diego, CA) in PBS and then stained for 30 minutes at room temperature with the following panel of antibodies: Ly6C PerCP-Cy5.5 (RB6-8C5, cat#45-5931-80; Thermofisher), Ly6G AF700 (1 A8, cat#561236; BD Bioscience), CD24 APC (MI/69, cat#562349; BD Bioscience), CD45 APC-Cy7 (30-F11, cat# 557659; BD Bioscience), CDl lb BV605 (MI/70, cat# 563015; BD Bioscience), CDl lc PE (HL3, cat# 557401; BD Bioscience), F4/80 PE-Cy5 (BM8, cat# 15-4801-82; Thermofisher), MHC II PE-Cy7 (M5/114.15.2, cat# 107629; Biolegend), and CD64 BV421 (X54-5/7.1, cat# 139309; Biolegend). After staining, cells were washed with PBS and resuspended in FACs buffer. Data was acquired with a BD Fortessa LRS flow cytometer using BD FACSDiva software (BD Bioscience). Compensation was performed on the BD LSRII flow cytometer at the beginning of each experiment. Data were analyzed using Flowjo. Gating strategy was adapted from Yu et al. (Yu et al., 2016).
[00123] Milliplex Mouse 32-Plex. Mouse serum and lung homogenate cytokine concentrations were determined using a multiplex immunoassay (cat# MCYTMAG-70K-PX32, Millipore) and analyzed using a BioPlex 200 (Bio-Rad, United States). Mouse cytokines and chemokines measured include: Eotaxin, G-CSF, GM-CSF, fFN-y, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-la, MIP-ip, MIP-2, RANTES, TNF, and VEGF. All protocols were performed according to manufacturer instructions.
[00124] Statistics. Survival curves were compared using the log rank (Mantel-Cox) test. Significant differences were defined at <0.05. Sepsis scores, weight changes, serum cytokine levels, and immune cell percentages from flow cytometry were compared using ANOVA followed by post hoc Student’s t-test. These statistical analyses were performed using Prism software (Graph Pad Prism 9).
Example 2: C. dubliniensis induces Gr-1+ cell-mediated protection against lethal challenge with C. albicans/E. coli but not LPS
[00125] We have previously established that immunization with live Cd 14-60 days prior to lethal challenge effectively induces trained innate protection against sepsis induced by polymicrobial IAI with several pathogenic Candida species and S. aureus (Lilly et al., 2018). Importantly, Cd is largely cleared by day 7 post-immunization with no evidence of further burden prior to challenge (Lilly et al., 2021). To determine whether this protection extends to IAI involving a common gram-negative bacterial abdominal pathogen, we first gave mice a lethal challenge of C. albicans and Escherichia coli strain ATCC 25922 (Ca/Ec). Similar to our murine model of Candida sp/Sa IAI, intraperitoneal inoculation of Ca/Ec results in synergistic lethality (Klaerner et al., 1997), whereas monomicrobial infections of each failed to result in mortality (FIG. 1A). To evaluate protection, mice were inoculated (immunized) i.p. with Cd and challenged 14 d later by i.p. inoculation with Ca/Ec. Mice were monitored for survival and sepsis scores using the modified M-CASS system (Mai et al., 2018) over a 10-day period. Results showed that prior immunization of mice with Cd resulted in 80% survival following Ca/Ec challenge compared with no survival in unvaccinated mice (p<0.0001) (FIG. IB). Unvaccinated mice exhibited severe morbidity including ruffling of fur, squinting and hunched posture prior to humane euthanasia indicated by high sepsis scores in 24-48 h, while immunized mice exhibited low sepsis scores with a limited number of sepsis/morbidity indicators over the 10-day period (FIG. 1C). We next examined the efficacy of the live Cd immunization in protecting against sepsis induced by E. coli- vd e LPS (LPS endotoxin serotype 0111 :B4) (10 mg/kg). ( ^/-immunized mice challenged 14 d later by i.p. injection with a lethal dose of LPS rapidly succumbed to sepsis, with 10% survival by 48 h post-injection which was not significantly different from the 20% survival observed in unvaccinated mice (FIG. ID) with high sepsis scoring in both groups (FIG. IE).
[00126] Based on the strong protection imparted by live Cd immunization against Ca/Ec challenge, we next tested the requirement for innate immune cell populations previously identified in other models (Gr-1+ leukocytes or macrophages) in mediating trained immune protection (Quintin et al., 2012; Lilly et al., 2018). For this, Cd immunized mice were treated with anti-Gr-l+ or isotype control antibodies every 48h beginning 48h prior to lethal challenge, or a single injection of clodronate-containing liposomes or empty liposomes 24h prior to lethal challenge with Ca/Ec, and monitored for mortality and sepsis scoring. Compared to the 80-100% survival in Cd immunized untreated or isotype antibody -treated control mice, Gr-1+ (Ly6C/Ly6G) cell depleted mice had high mortality rates (p<0.001) equivalent to unvaccinated control mice (FIG. 2). In contrast, cl odr onate-treated mice depleted of macrophages had survival rates similar to the empty liposome-treated mice and significantly higher than the unvaccinated control mice (P < 0.001) (FIG. 2A). Sepsis scores paralleled the mortality/survival results (FIG. 2B), including scores reflecting the modest reduction in survival in macrophage-depleted mice (-60%) (-5 at day 2 post-challenge) compared with control liposome treated mice (-1).
Example 3: C. dubliniensis immunization protects against zymosan induced sepsis with a similar role for Gr-1 + cells in protection
[00127] Because live Cd immunization induced protection in several models of fungal/bacterial IAI, we tested whether the protection extends to sepsis induced by zymosan, a cell wall extract of yeast that mimics human sepsis when administered i.p. at high concentrations by signaling via both TLR-2 and dectin-1 (Dillon et al., 2006; Lewis et al., 2016; Stortz et al., 2017). For these studies, mice were immunized with live Cd 14 d prior to i.p. challenge with a lethal dose of zymosan (lOOOmg/kg) and monitored for mortality and sepsis scores. Results showed that by day 3 post-challenge, the majority of unvaccinated mice had succumbed to sepsis (-20% survival). In contrast, the majority of immunized mice survived through day 10 (-90% survival; p<0.01) (FIG. 3A). Correspondingly, immunized mice exhibited mild signs of sepsis over the 10 day period, while unvaccinated mice displayed severe morbidity resulting in high sepsis scores within 24 h of lethal challenge (FIG. 3B).
[00128] To determine whether the cell populations necessary for effective Cd protection were the same as previously identified in both polymicrobial infection models, we repeated the challenge model in mice depleted of either macrophages or Gr-1+ cells using the same design. Results showed significantly reduced survival in immunized Gr-1+ cell depleted mice (60% survival) compared to the isotype antibody control immunized mice (100% survival) (p<0.05). Despite the uncharacteristically higher survival in Gr-1+ cell depleted mice, there was no statistical significance compared to unvaccinated mice (-40% survival). In contrast, survival in clodronate-treated and empty-liposome-treated immunized mice were similarly high (90% and 100%, respectively) (FIG. 4A). These effects are consistent with the sepsis scoring with Gr-1+ cell-depleted vaccinated mice having moderate-high scores similar to those of the unvaccinated mice (7-10 by day 2 post challenge), and mild sepsis scores (<5) in both groups of control- treated and clodronate-treated immunized mice throughout the observation period (FIG. 4B).
[00129] Taken together, while ^/-mediated protection was maintained in both polymicrobial Ca/Ec IAI and abiotic zymosan-induced sepsis models, none of the disclosed immunization strategies provided protection when mice were challenged with a lethal dose of LPS. However, there are distinct differences in LPS sepsis compared with the infection-based sepsis models disclosed herein. LPS-induced sepsis is an intoxication model, in which LPS is delivered as a single, large bolus of toxin, in contrast to the complex and repeated interactions between the microbial PAMPs and host PRRs that occur during polymicrobial lAI-mediated sepsis in mice and humans (Remick and Ward, 2005; Lewis et al., 2016). LPS administration in mice rapidly induces high circulating inflammatory cytokine levels, which peak much earlier than infection- induced sepsis (Rittirsch et al., 2007; Chen et al., 2014). Additionally, physiological effects are dose-dependent and large doses of LPS are required to induce responses similar to septic shock in mice (van der Poll, 2012; Chen et al., 2014). Therefore, the abiotic and live immunization strategies disclosed herein protect against sepsis resulting from infection, not septic shock induced by high doses of toxin exposure.
Example 4: Immunization with fungal H-glucan compounds results in protection against polymicrobial Ca/Sa IAI sepsis predominantly mediated by Gr-1+ cells
[00130] Previously published studies have identified abiotic agents as inducers of TII including fungal P-glucan, a component of the fungal cell wall (Quintin et al., 2014). We tested a variety of commercially available fungal P-glucan compounds for efficacy in inducing protection against polymicrobial IAI induced sepsis. Different formulations of fungal P-glucans are available as both soluble and particulate compounds that differ in receptor signaling capacity. For these studies, several preparations of purified P-glucan derived from S. cerevisiae were tested. The particulate P-glucan was either treated by incubation in alkaline buffer to increase solubility (modified) or left untreated and resuspended in sterile PBS (unmodified). We also tested a larger size P-glucan product, WGP (whole glucan particle) dispersible, which consists of hollow whole S. cerevisiae cell wall “ghosts”, which function as a dectin- 1 agonist (Goodridge et al., 2011). Mice were immunized by i.p. injection with 1 mg of the P-glucan preparations or 200 ug WGP dispersible and challenged 14 d later with lethal Ca/Sa IAI. While unmodified P-glucan and WGP were ineffective in inducing protection (-30% survival at day 4 post-challenge vs 0% survival in unvaccinated mice), modified (alkali-treated) P-glucan induced strong protection with 80% survival at day 10 post-challenge (p<0.05) (FIG. 5A). Even at higher doses, the untreated purified P-glucan showed no appreciable protection (data not shown). Lastly, we tested S. cerevisiae depleted zymosan (d-zymosan), which is treated with hot alkali solution to remove TLR signaling activity while retaining dectin-1 signaling (Ikeda et al., 2008). Mice were immunized by i.p. injection with 1 (day -14) or 2 (day -14, -7) doses of 1.2 mg d-zymosan and challenged with Ca/Sa IAI. Similar to our results with modified P-glucan, immunization with 1 or 2 doses of d-zymosan induced significant protection against sepsis with 90-100% of mice surviving through day 10 post-lethal challenge compared to 20% survival in unvaccinated mice (p<0.001 (FIG. 5B).
[00131] To assess the duration of protection with abiotic immunizations, we immunized mice with a single dose of d-zymosan or mod. P-glucan and challenged 24h or 72h post-immunization or immunized with d-zymosan as described previously and challenged 30, 60 or 90d postimmunization. Both abiotic immunizations were highly effective in protecting against death and morbidity (FIG. 9). We observed robust survival in as little as 24h post abiotic immunization with complete survival of mod. P-glucan immunized mice on both day 1 and day 3 post- immunization challenges (FIG. 9A). The overall survival for d-zymosan immunized mice was slightly but not significantly lower; we observed an 80% survival for both challenge days. The sepsis scores (FIG. 9B) were similar between both immunizations and on both challenge days. We observed low sepsis scores (<3) for each immunization group up to day 9 post-lethal challenge. These observed sepsis scores were significantly lower than the unimmunized mice (>6) who succumbed to the challenge by day 2 post-lethal challenge (p<0.001-0.0001). The immunized mice were sacrificed at study endpoint and spleens and peritoneal lavage fluid plated for pathogen burdens. Both abiotic immunizations challenged on day 1 or 3 post-immunization were able to clear both Ca and Sa from the peritoneal cavity (FIG. 9C) by study endpoint. Similarly, both abiotic immunizations on both challenge days 1 or 3 post-immunization cleared all pathogen from the spleen with the exception of one mouse in the d-zymosan immunization group that had been challenged 24h post-immunization (FIG. 9D). It is notable that the plated Sa burden in the mouse’s spleen was lower than the initial challenge dose. We also found that d- zymosan immunization conferred long term protection similar to that of Cd immunization (FIG. 10). We observed that mice challenged 30d or 60d post-immunization survived (FIG. 10A) significantly better than the unimmunized and age matched mice (p<0.05-0.01). The immunized mice challenged 90d post-immunization had the same overall survival percentage (40%) as the 60d mice but were not significantly different than unimmunized mouse overall survival. This is due to earlier mortality (days 2-3 post-lethal challenge) in both 90d and unimmunized mouse groups. Similarly, we observed the highest sepsis scores in the unimmunized mice with the majority reaching clinical end points by day 2 post-lethal challenge (FIG. 10B). We observed similarly high sepsis scores in the d-zymosan immunized mice challenged 90d postimmunization on days 1 and 2 post-lethal challenge. These mice reached clinical endpoints or recovered by day 3 post-lethal challenge with sepsis scores (~5 or less) more similar to the other immunization groups. We observed mild sepsis scores in the d-zymosan immunized mice challenged on days 30 and 60 post immunization with some variability in the 60d mouse group scores. Based on these results, both abiotic immunizations confer protection in as little as 24h post-immunization and d-zymosan immunization protection continues to 60 days postimmunization but wanes through 90 days post-immunization.
[00132] To determine whether the protection against IAI sepsis induced by abiotic P-glucan compounds was mediated by the same cell population that we had identified previously in Cd vaccination (i.e., Gr-1+ MDSCs), we performed cell depletions as described above and monitored survival and sepsis scores. Following immunization with modified P-glucan, we observed significant mortality in Gr-1+ cell depleted mice (10% survival) compared with isotype antibody-treated control mice (90% survival) (p<0.001) and delayed mortality in immunized macrophage-depleted mice (FIG. 6A) (p<0.001) compared to immunized control liposome- treated mice. Survival in the macrophage-depleted mice was significantly higher compared with unvaccinated mice (p<0.05) but not significantly different from survival in Gr-l+-depleted mice. These patterns are reflected in the results of sepsis scoring with both the Gr-l+-depleted mice and unvaccinated mice having high scores (range 10-18) by day 2 post-lethal challenge, and macrophage-depleted mice with modest scores (range 3-6) by day 3 post-lethal challenge (FIG. 6B) In contrast, the effects of cellular depletions in mice immunized with d-zymosan were more similar to Cd immunized mice; Gr-1+ cell depletion completely abrogated protection compared with isotype antibody control mice (0% vs 80% survival, respectively), while survival in macrophage-depleted mice was similar to that for empty liposome control mice (60% vs 90% survival, respectively) (FIG. 6C). Sepsis scores followed the same trends as mortality with unvaccinated and Gr-l+-depleted immunized mice having high sepsis scores compared to the vaccinated control mice, and macrophage-depleted immunized mice having overall scores similar to vaccinated mice (FIG. 6D). To determine whether protection induced by abiotic immunization resulted in reduced microbial burdens similar to previous studies (Lilly et al., 2021), peritoneal lavage fluid and spleens were analyzed for CFU levels at clinical endpoint for moribund mice or study endpoint for healthy animals. Immunization with either d-zymosan or alkali -treated P-glucan resulted in clearance of both C. albicans and S. aureus in most tissues at study endpoint compared with high levels in unvaccinated animals at clinical endpoint (FIGs.
8A-8B)
Example 5: Fungal H-glucan compounds induce protection against polymicrobial Ca/Ec IAI but not LPS sepsis
[00133] To determine if protection induced by abiotic P-glucan compounds against Ca/Sa sepsis also extends to the other models of sepsis, we tested their efficacy against Ca/Ec IAI and LPS-induced sepsis. For these experiments, mice were immunized once with modified P-glucan or twice with d-zymosan followed by lethal challenge of Ca/Ec i.p. or LPS 14 days later and monitored for survival and sepsis scoring. Similar to immunization with live Cd, both P-glucan compounds induced strong protection (75-100% survival through day 10) compared with unvaccinated mice (p<0.0001) (FIG. 7A). The protection in the immunized mice corresponded with low sepsis scores compared to unvaccinated mice (FIG. 7B) (p<0.0001). Similar to immunization with live Cd (FIG. 1), both P-glucan compounds failed to confer any significant protection against the LPS challenge (FIG. 7C). This was corroborated with the high sepsis scores in both vaccinated and unvaccinated mice (FIG. 7D).
Example 6: Variable requirement for IL-10 in trained innate protection against polymicrobial Ca/Sa IAI following biotic and abiotic immunization
[00134] We previously identified putative MDSCs as the main Gr-1+ effector immune cell in Cd and abiotic fungal P-glucan immunizations (Lilly et al., 2018). As one of the main effectors of MDSC-mediated immunosuppression, we examined the necessity of the anti-inflammatory cytokine IL-10 in the protection induced by our biotic and abiotic immunizations. For this, we immunized WT and IL-10'/_mice by i.p. injection with live Cd, mod. P-glucan or d-zymosan and challenged 7-14 d later with lethal Ca/Sa IAI. Mice were monitored for survival and sepsis scores using the modified M-CASS system (Mai et al., 2018) over a 10-day period. The survival percentage in mice immunized with live Cd (biotic immunization) was reduced significantly to 40% in IL-10'/_ mice when compared to immunized WT controls (FIG. 12A; p<0.01). Interestingly, we also observed significantly higher survival in the immunized IL-10'/_mice than in the unimmunized IL- 10"7" control group (10%, p<0.05). Furthermore, mortality of the immunized IL-10'/_mice was slightly delayed compared to unimmunized IL-10"7" mice. Similarly, the observed morbidity scores (FIG. 12B) of the Cd immunized IL-10"7" mice were higher (5-7) than the WT immunized mice (0-1) on days 2 and 3 post-lethal challenge and neither immunized group was significantly lower than the unimmunized IL-10"7" or WT mice (>7). These results suggest that IL- 10 plays a significant but partial role in (W-mediated protection.
[00135] In contrast, IL- 10 was not required for protection against lethal polymicrobial IAI induced following immunization with the abiotic immunizations (mod. P-glucan and d- zymosan). Mice immunized with d-zymosan demonstrated similar overall survival regardless of WT or IL- 10"7" genotype (FIG. 12C). We observed significantly higher survival percentages (90- 100%) in both immunized groups compared to their unimmunized counterparts (10%; p< 0.0001). A similar outcome was observed in the morbidity scores of the immunized mice (FIG. 12D) We observed significantly lower sepsis scores in both d-zymosan immunized groups of mice compared to the unimmunized groups (p<0.05-0.01). Interestingly, we did observe a slight but consistently elevated sepsis score (2-3) in the IL-10"7"mice immunized with d-zymosan when compared to WT immunized mice (0-1) but this trend was not significant. This data suggests no requirement for IL-10 in d-zymosan-mediated protection. Similar results were observed in IL-10" 7" mice immunized with mod. P-glucan (Fig 12D). We observed survival of 80-90% in the IL-10" 7" and WT mice immunized with mod. P-glucan (Fig 12E) , which was significantly higher than the overall survival in unimmunized mice regardless of genotype (10%; p<0.01-0.001). However, the observed morbidity scores (Fig 12F) were higher for the mod. P-glucan immunized IL-10'/_ mice. From days 1-3 post-lethal challenge, we observed mild-to-moderate morbidity scores (3-5) on average in the IL-lO'^mice immunized with mod. P-glucan compared to the WT immunized mice (0-1). This trend was not significant but the recorded sepsis scores for the IL-10'/_ immunized mice were also not significantly different from the sepsis scores of the unimmunized IL-10'/_ mice for 2 of these 3 days (day 2, p<0.05). These data seem to suggest that while IL- 10 is not required for mod. P-glucan-mediated protection against lethal Ca/Sa IAI, it may attenuate some of the morbidity from sepsis. This is especially true when comparing the IL- 10'/_ mice immunized with mod. P-glucan immunized to those immunized with d-zymosan.
[00136] To further investigate the variable requirement for IL- 10 in trained innate protection, we assessed pro- and anti-inflammatory cytokine concentrations in serum samples of abiotic immunized mice. For these experiments, mice were immunized with mod. P-glucan or d- zymosan, and then administered anti-Gr-1 or isotype control antibodies every 48h beginning 48h prior to lethal challenge with Ca/Sa and monitored for mortality and sepsis scoring. The results showed that measured serum levels of both inflammatory cytokines (IL-6, TNF, IL-ip, IL-la, IFNy) and anti-inflammatory IL-10 were higher in mice lacking Gr-1+ cells than in mice who received either of the abiotic immunizations but were not depleted of Gr-1+ cells (FIG. 13). Overall levels of all serum cytokines were higher in unimmunized control mice compared with control treated abiotic immunized mice with significantly higher levels observed in IL-6 levels observed (FIG. 13A). Levels of the hallmark pro-inflammatory cytokine IL-6 were significantly higher in serum samples from all Gr-1+ cell depleted abiotic immunized mice compared with control treated immunized mice (FIG. 13A). Immunized mice depleted of Gr-1+ cells had serum concentrations of -35000 pg/mL compared to virtually undetectable IL-6 in immunized mice who received isotype antibody (p<0.0001). We observed no differences in IL-6 levels between mod. P-glucan groups who received clodronate or liposomes. Other pro-inflammatory cytokines (IFN-y, TNF, IL-ip, IL-la) were also significantly higher in the d-zymosan immunized, Gr-1+ cell depleted mice when compared to either isotype treated group (FIGs. 13B-13E; p<0.01- 0.0001). Similarly, concentrations of these serum cytokines were higher in mod. P-glucan immunized Gr-1+ cell depleted mice compared with control treated mice, although these differences were only significant for IFN-y and IL-ip. We observed no differences in these cytokines in either abiotic immunization group when they received clodronate or liposomes.
There was a slight but insignificant increase in TNF when comparing mod. P-glucan immunized mice depleted of macrophages and liposome treated mice. Surprisingly, we measured higher levels of the anti-inflammatory cytokine IL-10 in abiotic immunized Gr-1+ cell depleted serum compared to isotype control treated immunized mice with significant differences only observed in d-zymosan immunized mice (p<0.05) (FIG. 13F). We saw no differences in serum IL-10 levels in any abiotic immunized mice depleted of macrophage when compared to liposome control groups. Overall, these results indicate that Gr-1+ cells are necessary for limiting pro- inflammatory cytokines in abiotic immunizations with fungal cell wall compounds. However, a reciprocal reduction in IL- 10 levels does not necessarily correlate with the abrogation of protection observed in Gr-1+ cell depleted mice, which may partially explain the phenotype observed in IL-1 O'" mice.
Example 7: PRR Signaling Pathways Differ in response to Biotics v.s Abiotic Immunization [00137] In previous studies, mice immunized with biotic (live Cd) and abiotic (d-zymosan or mod. P-glucan) products exhibited differences in their requirements for Gr-1+ cells and macrophages. These previous results combined with the differences in IL- 10 requirements/cytokine profiles between immunizations prompted us to examine the innate immune recognition and signaling pathways involved in protection using PRR reporter cell lines. We incubated HEK-Blue PRR reporter cells (Dectin-la, Dectin-2, TLR2 and TLR4) with live Cd (1 : 10 dilution), d-zymosan (10 pg/mL) or mod. P-glucan (10-100 pg/mL) to examine their respective PRR signaling pathways (FIG. 14). Unmodified zymosan or LPS served as positive controls and PBS as a negative control for the assays. The results demonstrated that both d- zymosan (10 pg/mL) and mod. P-glucan (100 pg/mL) signal through Dectin-la (FIG. 14A), with both abiotic products inducing significantly higher signals than the negative control (PBS; p<0.0001) and d-zymosan being comparable in magnitude to the unmodified zymosan positive control (10-100 pg/mL). As a control, we included unmodified particulate P-glucan (100 pg/mL) from S. cerevisiae which also signaled strongly through Dectin-la, as reported in other studies (p<0.0001). Conversely, live Cd signaled weakly through Dectin-la, and was not significantly different from PBS. By contrast, live Cd demonstrated robust signaling through the Dectin-2 (FIG. 14B). In fact, live Cd was the only immunization that induced a signal significantly higher than the negative control in the Dectin-2 reporter cells (p< 0.0001); however, the Ct/-induced signal was still lower than the zymosan positive control. For the TLR2 reporter cells (FIG. 14C), among the immunization, only exposure to mod. P-glucan (10 pg/mL) resulted in any significant signal compared to PBS (p<0.0001). Interestingly, the higher concentration of mod. P-glucan
(100 pg/mL) had diminished signaling similar to the other immunizations tested and the negative control. This may suggest that there is an “ideal” concentration range for mod. P-glucan signaling rather than a simple dose-response. Unsurprisingly, none of the tested immunizations showed any significant signaling through TLR4 (FIG. 14D); the abiotic fungal compounds used for immunization were processed to remove LPS prior to administration. These differences in recognition by PRRs likely translate to variances in TTI responses and may help explain the differential requirement for cell types and cytokine requirements.
Example 8: C. dubliniensis Induces Protection Against Lethal Challenge with C. albicans/S. aureus through CARD9 signaling pathways
[00138] To test the requirement for PRR signaling pathways in trained innate protection induced by biotic live Cd immunization, we immunized mice lacking the MyD88 adaptor protein, Dectin- 1 receptor or the CARD9 adaptor protein. Mice were monitored for survival and morbidity for 10 days following lethal challenge. The in vivo data demonstrated a clear role for only the CARD9 adaptor protein in Cd immunization-mediated protection. In MyD88'/_mice (FIG. 15A), we observed equally high overall survival in (%/-immunized MyD88'/_mice as in /-immunized WT mice (100% survival in both groups), and survival percentages among immunized mice were significantly higher than in the unimmunized mice of both genotypes (p<0.01-0.001). We observed a characteristic low survival (0-20%) in unimmunized MyD88'/_ mice and WT mice, with the MyD88'/_mice all succumbing by day 9 post-lethal challenge.
Similarly, in the morbidity scores (FIG. 15B), we consistently observed very mild sepsis scores (0-1) in both groups of immunized mice; these sepsis scores were lower than their unimmunized counterparts, though only the WT mice had significant differences in their scores on days 1 and 3 post-lethal challenge (p< 0.01 and 0.05, respectively).
[00139] Consistent with the in vitro data, the recorded survival percentages were similar for all Cd immunized mice regardless of the presence of Dectin- 1 signaling (FIG. 15C). We observed complete survival in the Cd immunized Dectin-l'7' and WT mice (100% survival) and both groups had significantly higher survival percentages than either unimmunized group (p<0.05). The lack of the Dectin-1 receptor also had no effect on /-mediated morbidity during IAI (FIG. 15D). Again, we observed mild sepsis scores (0-1) in both groups of immunized mice. Observed sepsis scores in Cd immunized Dectin- 1'/_ mice were significantly lower on day 1 post- lethal challenge than the sepsis scores in the unimmunized Dectin- 1'/_ mice (p<0.05). Similarly, observed sepsis scores in Cd immunized WT mice were also significantly lower on days 2 and 3 post-lethal challenge when compared to unimmunized WT mice (p<0.01-0.001). These results confirmed our reporter cell data that neither Dectin- 1 nor TLRs are necessary in Cd immunization recognition and signaling.
[00140] Unlike MyD88 and Dectin- 1, we did observe a critical role for CARD9 in both survival (FIG. 15E) and sepsis scores (FIG. 15F). While WT immunized mice had a robust survival (100%) that was significantly different from the unimmunized WT mice (20%; p<0.001), we observed a low overall survival (30%) in the immunized CARD9'/_ mice, demonstrating a loss of Gf-mediated protection in these mice. This survival was not significantly different from the unimmunized CARD9'/_mice (10% survival). Furthermore, in terms of morbidity, we observed virtually identical sepsis scores the CARD9'/_ mice, regardless of immunization status while there were significantly lower sepsis scores in the WT immunized mice than in their unimmunized WT control group (p<0.05). This data supports a role for CARD9 signaling in Cd immunization, likely through Dectin-2 signaling as noted in the in vitro reporter cell experiments, while MyD88 and Dectin- 1 signaling are dispensable.
Example 9: Immunization with Modified Fungal H-Glucan Induces Protection Against Lethal Challenge with C. albicans/S. aureus Independent of CARD9, Dectin-1, or MyD88-dependent Signaling
[00141] To examine the role of PRR signaling in protection induced by abiotic mod. P-glucan, we immunized mice i.p. as mentioned previously and challenged 7 days later with lethal Ca/Sa. Based on the in vitro experimental data, we hypothesized that both Dectin-1 (and subsequently CARD9) as well as MyD88 would play a role in mod. P-glucan immunization signaling. Conversely, we saw high survival rates (70-80%) in the mod. P-glucan-immunized mice regardless of the presence or absence of the MyD88 adaptor protein (FIG. 11 A). Survival percentages were significantly higher in both immunized mouse groups (MyD88'/_ and WT) than in the unimmunized mouse groups (p<0.05-0.01). Similarly, we observed high sepsis scores (FIG. 11B) in the unimmunized MyD88'/_ and WT mice when compared to both immunized mouse groups, although this trend did not reach significance. We recorded variable but generally mild scores in both immunized mouse groups, averaging <4, throughout the course of the study. These data indicate that MyD88 is not required for mod. P-glucan-mediated protection but may still play some role in attenuating morbidity.
[00142] Similarly, we observed complete survival (100%; FIG. 11C) in immunized mice regardless of the presence or absence of Dectin-1 receptor signaling, and both mod. P-glucan immunized groups showed significantly higher survival than their unimmunized control groups (p<0.01-0.001). We observed low survival percentages (0-10%) in unimmunized Dectin-l'7' and WT mice with the majority of both groups reaching clinical endpoints quickly (i.e., by day 2 post-lethal challenge). We also observed similar sepsis scores (FIG. 11D) regardless of mouse genotype, and differences in sepsis scores were fully dependent on immunization status. We saw minimal morbidity in both groups of mod. P-glucan immunized mice, where observed scores remained low (<1) throughout the study. We also observed significantly lower sepsis scores in the immunized Dectin- r/_ mice compared to the unimmunized Dectin- 1'/_ mice on day 2 (p<0.01) and 3 (p<0.0001) post-lethal challenge. We saw significantly lower morbidity scores from day 1 post-lethal challenge onward for WT mod. P-glucan immunized mice compared to WT unimmunized mice (p<0.01-0.0001). These results indicate a completely dispensable role for Dectin- 1 signaling in mod. P-glucan -mediated protection against lethal Ca/Sa.
[00143] We observed similar results for the mod. P-glucan immunizations and their requirement for CARD9 signaling in protection. In experiments with CARD9'/_ mice, mod. P- glucan-mediated protection was independent of CARD9 signaling with regards to both survival (FIG. HE) and in morbidity scores (FIG. HF). Similar to the Dectin-l'7' mice, we observed high overall survival (80%) in the mod. P-glucan immunized CARD9 mice, very similar to the Dectin- 1'/_ mouse data, although we saw slightly lower survival percentages in immunized CARD9'/'mice compared to WT immunized mice (100%). Documented survival percentages in both immunized CARD9'/" and WT groups were significantly higher than their unimmunized counterparts (0-10%; p<0.01 ). The observed sepsis scores (FIG. HE) were almost identical to those observed in the Dectin-l'7' mice. We saw mild morbidity scores (0-1) in both mod. P- glucan immunized groups with the immunized CARD9'/'mice having a slight peak in sepsis scores on day 6 post-lethal challenge (0-2). We observed significantly lower scores for mod. P- glucan immunized groups (CARD9'/_ and WT) compared to the unimmunized groups of their respective genotypes on day 1 (p<0.01-0.0001) and WT immunized mice had significantly lower recorded sepsis scores on day 2 (p<0.0001). The unimmunized CARD9'/' mice had all reached clinical endpoints before day 2 post-lethal challenge so there were no scores to compare to the mod. P-glucan immunized CARD9'/'mice. The results from these studies indicate a nonessential role for CARD9 signaling in mod. P-glucan-mediated protection. Remarkably, there was no clear necessity for any of the PRRs (Dectin-1) or adaptor proteins (MyD88 and CARD9) tested in these mouse studies for mod. P-glucan immunizations. Example 10: C. dubliniensis induces protection against lethal chailense with H1N1 Influenza [00144] With the efficacy of live Cd immunization in inducing trained innate protection in various sepsis models, we tested whether the protection also extends to viral respiratory infections. We chose to test immunization against H1N1 influenza challenge as the pathology of influenza infections is significantly attributable to host inflammation, similar to sepsis. For these studies, mice were immunized with live Cd (1.75 x 107 CFU, i.p.) 14 d prior to challenge with a lethal dose of H1N1 influenza (oropharyngeal aspiration, 15 PFU per mouse) and monitored for mortality, weight loss and sepsis scores. We observed robust protection (80% survival) in Cd- immunized mice (FIG. 16A), which was significantly higher than unimmunized mice. Similarly, we observed only moderate morbidity in immunized mice (FIG. 16B) over the course of the study and these mice maintained sepsis scores <6, which were significantly lower than the control mice from day 10 post-lethal challenge onwards (p<0.01-0.0001). In contrast, the unimmunized mice became extremely moribund by days 9-10 post-lethal challenge, and all reached clinical endpoints by day 13 post-lethal challenge (p< 0.001). We recorded high sepsis scores among unimmunized mice until they succumbed to challenge. We observed similar weight loss in both immunized and unimmunized mice until day 12 post-lethal challenge. However, the weights of Cd immunized mice rebounded at later time point, while unimmunized mice were unable to recover their weight even after clearing the virus (FIG. 16C). These data suggests that Cd immunization protects against influenza morbidity and mortality.
[00145] The efficacy of the Cd immunization against influenza infection led us to next explore the mechanisms involved. To determine whether the cell populations necessary for effective Cd protection against influenza were similar to those previously identified in our other sepsis models, we repeated the above experiments in mice depleted of circulating macrophages or Gr-1+ cells. For these depletion studies, Cd immunized mice were administered i.p. anti-Gr-1 antibody to deplete Gr-1+ cells. To deplete circulating macrophages, mice were administered i.p. clodronate liposomes. To deplete local phagocytic cells in the lungs (i.e., AMs), mice received intranasal (i.n.) clodronate liposomes. Unimmunized mice that were not depleted of cells and served as negative controls while IgG isotype antibody or control liposomes served as sham depletions. Contrary to the protection seen in Ca/Sa sepsis models, the results here showed that depletion of neither circulating macrophages nor Gr-1+ cells had a significant effect on mouse survival (FIG. 17A). We observed high survival rates (80%) in the Cd immunized mice treated with aGr-1 or isotype antibody while unimmunized mice reached clinical endpoints by day 9 post-lethal challenge. Immunization protection was also represented in mild morbidity scores (FIG. 17B) recorded for both Cd immunized groups (aGr-1 and isotype control antibody) whereas we observed consistently high sepsis scores (>7) from day 7 post-lethal challenge in the unimmunized mice. We also observed rebounding average weights (FIG. 17C) in both immunized mouse groups regardless of depletion or sham treatment, but unimmunized mice did not recover from influenza challenge. The results were similar overall for circulating macrophage depleted mice (FIGs. 17D-17E). We observed robust survival percentages (FIG. 17D). In the Cd immunized i.p. clodronate liposome mice (90%) which was slightly but not significantly higher than the Cd immunized i.p. control liposome mice (70%). We observed no survival to study endpoint in the unimmunized mouse group and all mice were humanely euthanized by day 11 post-lethal challenge. The measured morbidities (FIG. 17E) followed similar trends to the previous Cd immunization experiments. We observed mild scores in the Cd immunized i.p. clodronate treated mice (<4) and slightly higher sepsis scores in the Cd immunized i.p. liposome treated mice (<6). We observed moderate-to-high (>7) sepsis scores in the unimmunized mouse group from day 7 until all mice reached clinical endpoints by day 11 post-lethal challenge. We also observed recovery in average weights (FIG. 17F) in the Cd immunized i.p. clodronate or control liposome treated mice similar to the observed rebounding weights of immunized aGr-1 treated mice (FIG. 17C). Overall, we observed high survival rates and mild morbidity in all Cd immunized mouse groups but not in the unimmunized mice. We also observed recovery in average weights in the immunized mice, whereas the unimmunized control mice succumbed to infection. These results suggest that circulating macrophages and monocytes, as well as Gr-1+ cells, are not required for ^/-mediated protection.
[00146] The efficacy of clodronate liposomes in depleting resident or circulating macrophage populations varies with delivery method. While we and other have demonstrated that i.p. delivery depletes circulating macrophages, this method of administration has no effect on lung resident macrophages (Hogg et al., 2021; Huang et al., 2018). Because lung resident AMs are critical in clearing virally infected cells and managing inflammation during influenza infection, we hypothesized that they may play a significant role in ^/-mediated protection. To target deplete lung resident macrophages, we next directly administered clodronate to the respiratory tract via i.n. administration, similar to previous studies (Huang et al.. 2018). Our results demonstrated that depletion of lung resident macrophages via i.n. clodronate liposome delivery abrogated /-mediated protection against mortality to levels equivalent to unimmunized mice (0%; FIG. 17G). We observed higher survival rates (40%) in the Cd immunized control liposome (i.n.) treated mice, but this difference was not significant. Additionally, we recorded high morbidity scores (8-10; FIG. 17H) by day 8 in the immunized i.n. clodronate liposome mice, which were significantly higher scores overall when compared to Cd immunized control liposome i.n. mice (p<0.01). We recorded moderate sepsis scores in the immunized control liposome-treated mice (5-7) at their peak. Moreover, the average weight changes followed similar trends (FIG. 171); we observed recovery of average weight (80% of their starting weight) in the Cd immunized control liposome i.n. mice alone, while the unimmunized and AM-depleted mice all reached clinical endpoints by days 10 and 12, respectively. These data indicate a role for tissue resident AMs in (^/-mediated protection against lethal H1N1 influenza infection although we must confirm depletion via flow cytometry.
[00147] It is anticipated that alkali-treated fungal P-glucans will also confer comparable protection against lethal challenges with the H1N1 Influenza model as described above in view of the similar therapeutic and phenotypic effects observed in animals immunized with alkali- treated fungal P-glucans and live C. dubliniensis.
Example 11: C. dubliniensis immunization improves viral clearance
[00148] To better understand the mechanisms through which Cd immunization mediated the protection observed during lethal influenza infection, we extracted whole lung RNA to quantify the relative expression of influenza viral RNA as a measure of viral load/clearance on days 3 and 7 post-lethal challenge. We found that viral RNA was undetectable at both 3 and 7 days post- lethal challenge in Cd immunized mice after 40 rounds of PCR amplification (FIG. 18; undetected data points not shown on graph). Overall, the relative expression of viral RNA was higher on day 7 than on day 3 in unimmunized mice although less mice had detectable RNA overall. Conversely, we observed detectable viral RNA in 3 of 5 unimmunized mice on day 3 post-lethal challenge and 2 of 5 unimmunized mice on day 7 post-lethal challenge. These data suggest that Cd immunized mice have an enhanced ability to clear the influenza virus when compared to unimmunized mice.
Example 12: C. dubliniensis immunization protection involves macrophage activation and anti-inflammatory cytokines
[00149] To assess the changes in immune cell populations induced by Cd immunization, lung immune cell populations were isolated from immunized and unimmunized mice at 3- and 7-days post influenza challenge and stained for analysis via flow cytometry. The flow panel and gating strategy was adapted from Yu et al. (Yu et al., 2016) and we analyzed the following cells populations: IM (F4/80+MHC II+), AM (CD64+CD1 Ic+CDl lb"), monocytes (Ly6G“CD64+CDl Ib+MHC II-), PMNs (Ly6G+), NK (CD64-MHC H-CDl lb+), eosinophils (CD1 lb+MHCII-CD24+), total macrophages (CD64+MHC 11+ or CD64+CD1 lb+), T cells (CD1 lc-CD24+MHC II-), B cells (CD1 lc-CD24+MHC 11+) and DCs (CD64+/-MHC II+Ly6G-). Based on the significant decrease in mortality among the immunized but AM- depleted mice, we also assessed lung macrophage populations after lethal challenge. We detected a general trend toward higher percentages of lung macrophages overall in Cd immunized mice. From study day 3 to day 7, we observed a significant increase in macrophage percentages (Fig 19A) in the Cd immunized mice (p< 0.05). On day 7, we also observed significantly higher percentages of macrophages compare to unimmunized mice (p<0.01). In both groups, we observed that AM percentages were elevated on study day 3, while interstitial macrophages (IMs) were elevated on study day 7. This transition is typical of highly pathogenic PR8 (H1N1) influenza which has been shown to deplete AMs in the lungs of (unimmunized) infected mice significantly from days 3-7 post-challenge with populations recovering by day 9 (Ghoneim et al. 2016). IMs are also depleted early in influenza infection although to a lesser degree and expand rapidly in the lung compartment approximately 1-week post-challenge to replace the depleted population of AMs. Cd immunized mice had higher percentages of both AMs (-12%) and IMs (-17%), although their levels did not reach significance when compared to unimmunized mice (7% and 12%, respectively). Overall, we observed significantly higher percentages of all macrophages, including AMs and IMs, in immunized mice from day 3 to 7 (p< 0.0001). We observed a similar increase in macrophages in unimmunized mice, but this increase was only significant for the IMs (p< 0.001).
[00150] We observed a decrease in both T and B cell percentages between days 3 and 7 postchallenge in the unimmunized mice (FIGs. 19B-19C), while percentages remained relatively low but consistent in immunized mice. The percentage of B cells was significantly lower in the immunized mice (-4%) when compared to unimmunized controls (10%) on day 3 post-lethal challenge (p<0.01). In general, we observed higher percentages of T cells (FIG. 19C) than B cells in the lungs of all mice for both days tested. We observed slightly higher T cell percentages in unimmunized mice (-44%) than immunized mice (-35%) on day 3 post-lethal challenge. Furthermore, we observed a significant decrease in T cell percentages in unimmunized mice, from 44% to less than 30%, over the 4-day span (p< 0.01). This supports our prior data that Cd immunization doesn’t rely on adaptive immune responses and is dependent upon innate cells to mediate protection.
[00151] To further characterize the inflammatory changes induced by Cd immunization, lung tissue homogenates and serum were collected from immunized and unimmunized control mice for multiplexed cytokine analyses at 3- and 7-days post influenza challenge. Cytokines analyzed included: IFNy, TNF, IL-6, IL-1, IL-10, IL-17, IL-2, IP10, MIG, MCP-1, and MIP-la (Fiore- Gartland et al., 2017; Hayney et al., 2017; Peiris et al., 2009; Tisoncik et al., 2012). We observed that Cd immunized mice had significantly increased levels of anti-inflammatory cytokines (IL- 10) and cytokines associated with anti-viral responses, including macrophage activation (IFNY), and pro-inflammatory cytokines (IL- lb, IL- la, IL-6, TNF) in our multiplexed cytokine analysis (FIG. 20) (Peiris et al., 2009; Tisoncik et al., 2012). Specifically, we observed significantly elevated levels of IL-10 in the lungs of Cd immunized mice when compared to unimmunized controls at day 7 post-challenge (p< 0.05). In the serum, we also observed higher levels of IL-10 on both 3 and 7 days in Cd immunized mice when compared to unimmunized mice (p<0.001 and p< 0.01, respectively; FIG. 20A). We also observed dramatically higher levels of IFNy (FIG. 20B) in the lungs of immunized mice (9500-11000 pg/mL) on days 3 and 7 post-lethal challenge compared to unimmunized mice (p<0.01-0.001). On day 3, serum cytokine levels were also significantly elevated in Cd immunized mice compared to unimmunized mice (p<0.01). IFNy is critical in regulating immune responses, both innate and adaptive, influenza infections. Mice deficient in IFNy are unable to clear influenza virus within 7 days and had significantly lower survival (Bot et al., 1998). We also observed the highest levels of both IL-ip and IL-la levels in the lungs (FIGs. 20C-20D) on study day 3 in unimmunized mice (-200 pg/mL and 480 pg/mL, respectively); these levels were significantly higher in unimmunized mice than in Cd immunized mice (-100 pg/mL and 200 pg/mL, respectively; p<0.05). Both IL- ip and IL-la cytokine levels trended highest early (day 3) for unimmunized mice and was significantly higher than observed levels of these cytokines on day 7 post-lethal challenge (p<0.05). The levels of IL-ip and IL-la were consistent in the lungs and serum of Cd immunized mice across both collection days, which may be indicative a level of controlled inflammation that is absent in the unimmunized mice. IL-1 is released early in influenza infections, followed by IL-6, and is linked to significant immunopathology in the tissues (Tisoncik et al., 2012). Conversely, IL-6 levels (FIG. 20E) were more variable between on both collection days and between both groups with no significant differences or clear trends. The highest levels of IL-6 were seen in the serum of immunized mice at day 7 post-lethal challenge (-440 pg/mL). High levels of IL-6 are secreted in response to tissue damage and virus recognition in influenza infections (Scheller et al., 2011). IL-6 is pleiotropic and immunoregulatory; it can be involved in virus clearance or T cell activation and functions but beneficial or harmful effects are dependent on synergism other cytokines (e.g., IL-1, IL-7 and IL-15) (Cox et al., 2013). We observed consistently low levels of TNF and IL-17 in both groups on both collection days, both locally and systemically (FIGs. 20F-20G). Overall, these results indicate that prior Cd immunization resulted in high levels of anti-inflammatory cytokine IL- 10 and anti-viral cytokine IFNy while we observed higher levels of inflammatory cytokines in their unimmunized counterparts following lethal influenza challenge.
[00152] In addition to anti-viral and anti-inflammatory cytokines, Cd immunization also resulted in increased the levels of chemokines (IP 10, MIG) involved in the recruitment of innate immune cells (including macrophages and monocytes). IP 10 is principally a chemokine involved in innate immune responses and is mainly secreted by macrophages and dendritic cells. We observed higher levels of IP10 in the serum of immunized mice (almost double the levels in unimmunized mice) on both days 3 and 7 post-lethal challenge (p<0.01 for both days) (FIG.
20H). Likewise, we observed slightly higher levels of MCP-1 and MIP-la in immunized mice in the lung homogenates, but the results were somewhat variable (FIGs. 201-20 J). We did not observe significant differences in either cytokine (MCP-1 and MIP-la) in the lung homogenate levels on either day although levels were slightly higher in Cd immunized mice; however, there were significantly higher levels of MIPla in the serum of Cd immunized mice on day 7 post- lethal challenge when compared to unimmunized controls (p<0.05). Both chemokines are involved in monocyte recruitment and likely have effects later in infection to replace lung resident macrophages damaged by inflammation (Chen et al., 2020; Fiore-Gartland et al., 2017). We also observed higher levels of MIG (or CXCL9; approximately 550-560 pg/mL), a cytokine induced by IFNY signaling, in the immunized mice on both days in the lungs (FIG. 20K) compared to unimmunized mice (<250 pg/mL; p< 0.05 and p<0.01, respectively). MIG amplifies IFNY signaling and is secreted by macrophages, monocytes and other antigen presenting cells. Likewise, both GCSF and MCSF trended slightly higher in immunized mice compared to unimmunized mice, but these differences did not reach significance (data not shown). Overall, anti-inflammatory (IL- 10), anti-viral (IFNY) and innate immune activation cytokines were consistently upregulated in the immunized mice. This may indicate that the local immune cells in Cd immunized mice are poised to respond more quickly and efficiently against a heterologous viral challenge than those in unimmunized mice. By contrast, we observed consistently higher inflammatory cytokines (i.e., IL-lb, IL-la, IL-6, TNF) and an ineffective immune response to viral challenge in unimmunized mice.
[00153] Overall, anti-inflammatory, anti-viral and innate immune activation cytokines were consistently upregulated in the Cd immunized mice. This may indicate that the local immune cells in Cd immunized mice are poised to respond more quickly and efficiently against a heterologous viral challenge than those in unimmunized mice. By contrast, we observed consistently higher inflammatory cytokines and an ineffective immune response to viral challenge in unimmunized mice. These elevated inflammatory cytokines in unimmunized mice exacerbate infection and result in high morbidity scores and mice reach clinical endpoints prior to study end.
[00154] These results demonstrate that immunization with live Cd or abiotic fungal compounds confers protection against polymicrobial sepsis of broad microbial origin, including both Gram" and Gram+ bacterial pathogens, as well as fungal pathogens.
EQUIVALENTS
[00155] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[00156] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[00157] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
[00158] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
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Claims

WHAT IS CLAIMED IS
1. A method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of an alkalinized fungal P- glucan extract.
2. The method of claim 1, wherein the alkalinized fungal P-glucan extract is derived from Saccharomyces cerevisiae, optionally wherein the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l,3) backbone via P-(l,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol.
3. The method of claim 1 or 2, wherein the alkalinized fungal P-glucan extract is obtained by treating purified P-glucan derived from a fungus with an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide.
4. The method of claim 3, wherein the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising 0.1 M borate buffer at a pH of about 9.8.
5. The method of claim 4, wherein the purified P-glucan is treated with the alkali solution for about 1 to about 12 hours at room temperature.
6. The method of claim 3, wherein the alkalinized fungal P-glucan extract is obtained by treating the purified P-glucan with an alkali solution comprising an alkali-metal hydroxide or an alkali earth-metal hydroxide having a concentration of from about 0.01 N to about 10. ON at a pH of about 7 to about 14.
7. The method of claim 6, wherein the purified P-glucan is treated with the alkali solution for about 1 to about 3 hours at a temperature of from about 4° C to about 121° C.
8. The method of claim 6 or 7, wherein the alkali-metal hydroxide is NaOH or KOH or wherein the alkali earth-metal hydroxide is Mg(OH)2 or Ca(OH)2.
9. The method of any one of claims 1-8, wherein the alkalinized fungal P-glucan extract is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
10. A method for preventing or treating infection-induced sepsis in a subject in need thereof comprising administering to the subject an effective amount of live wild-type Candida dubliniensis.
11. The method of claim 10, wherein the live wild-type Candida dubliniensis is Candida dubliniensis strain Wu284.
12. The method of claim 10 or 11, wherein the live wild-type Candida dubliniensis is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.
13. The method of any one of claims 1-12, wherein the infection-induced sepsis is caused by a polymicrobial infection.
14. The method of any one of claims 1-13, wherein the infection-induced sepsis is caused by a viral infection, a fungal infection, or a bacterial infection.
15. The method of claim 14, wherein the viral infection is caused by a virus selected from the group consisting of HIV, influenza virus, Ebola virus, chicken pox virus, Hepatitis B virus, HPV, measles virus, paramyxovirus, norovirus, rubella virus, Rous Sarcoma Virus, rabies virus, and rotavirus.
16. The method of claim 14, wherein the bacterial infection is caused by grampositive bacteria or gram-negative bacteria.
17. The method of claim 16, wherein the gram-negative bacteria is selected from the group consisting of Enter obacter spp., Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citr obacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp, and Vibrio spp.
18. The method of claim 16, wherein the gram-positive bacteria is selected from the group consisting of Bacillus spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., Mycobacterium spp., Corynebacterium spp. and Clostridium spp.
19. The method of claim 14, wherein the fungal infection is caused by a fungus selected from the group consisting of Candida spp., Cryptococcus spp., Coccidioides spp., Histoplasma spp., Blastomyces spp., and Pneumocystis spp.
20. The method of any one of claims 1-19, wherein the subject is an immunocompromised subject, a pediatric subject, a geriatric subject, or an adult subject.
21. A kit for treating or preventing infection-induced sepsis in a subject in need thereof comprising a fungal beta-glucan, an alkalinization agent and instructions for use, wherein the fungal beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l,3) backbone via p~( 1 ,6) linkages, and has a range of average molecular weight of about 200,000 g/ mol.
22. The kit of claim 21, wherein the instructions for use comprise instructions for alkali-treating the fungal beta-glucan prior to immunizing the subject.
23. The kit of claim 22, wherein the alkali-treated fungal beta-glucan is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral or intranasal administration.
24. The kit of any one of claims 21-23, wherein the alkalinization agent is an alkali solution comprising a borate buffer, an alkali-metal hydroxide, or an alkali earth-metal hydroxide.
PCT/US2023/068078 2022-06-08 2023-06-07 COMPOSITIONS INCLUDING CANDIDA DUBLINIENSIS AND ALKALINIZED FUNGAL β-GLUCANS FOR PROTECTION AGAINST INFECTION-INDUCED SEPSIS WO2023240146A2 (en)

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