Patent Application
20230076434
The present invention, in some embodiments thereof, is in the field of microbiology, microbiome, and related applications.
The microbiota has been recently shown to have major effects on human health. The composition of the microbiota changes in health and diseases states. Studies have shown that the microbiota composition can have beneficial effects on patients. These include both effects on the health state and on response to therapy. Recently, the microbiota as a whole, has been implicated to clinical practice, in forms of fecal transplantations (FMT). The first clinical indication, and the most successful one to date, was FMT, to treat Clostridium difficile diarrhea. However, currently, there are many clinical trials of FMT treatment for a variety of indication. Important to note that the common fecal source is a healthy human donor. One major challenge in FMT treatment is the microbiota composition of the transplanted feces. In some cases, even traces of unwanted microbes can disqualify the fecal transplantation. A method for eliminating unwanted microbes from a fecal composition, so as to requalify feces for transplantation (e.g., by FMT) in patients, is greatly needed.
According to a first aspect, there is provided a method for isolating a target microorganism from a sample comprising a plurality of microorganisms, the method comprising contacting the sample with an antibody having specific affinity to the target microorganism, wherein the antibody being produced by immunizing a host organism using a selected target microorganism, wherein the selected target microorganism is selected by contacting a fraction of the sample with one or more polynucleotide molecules each having specific affinity to one target microorganism, and determining the presence of the target microorganism in the fraction of the sample, thereby isolating a target microorganism from the sample comprising a plurality of microorganisms.
According to another aspect, there is provided a method for isolating a target microorganism from a sample, the method comprising the steps of: (a) providing a fraction of the sample comprising a plurality of microorganisms; (b) contacting the fraction of the sample with one or more polynucleotide molecules each having specific affinity to one target microorganism, and determining the presence of the one or more target microorganism in the fraction of the sample; (c) selecting the one or more target microorganisms determined to be present in the fraction of the sample and immunizing a host organism using one of the selected target microorganisms, thereby producing an antibody having specific affinity to the one selected target microorganism; and (d) contacting the sample with the produced antibody having specific affinity to the one selected target microorganism, thereby isolating a target microorganism from the sample.
According to another aspect there is provided a sample obtained by the herein disclosed method.
According to another aspect, there is provided a composition comprising the herein disclosed sample and a pharmaceutically acceptable carrier.
In some embodiments, the microorganism is selected from the group consisting of: bacteria, fungi, archaea, protozoa, and algae.
In some embodiments, the selected microorganism is a specific species or a specific strain of a microorganism.
In some embodiments, the microorganism is a pathogenic microorganism.
In some embodiments, the microorganism is a probiotic microorganism.
In some embodiments, contacting comprises contacting the sample with a plurality of polynucleotide molecules each having specific affinity to one target microorganism.
In some embodiments, the sample is selected from the group consisting of: a sample derived from a subject, a soil sample, and a water sample.
In some embodiments, the sample derived from a subject is a stool sample of the subject.
In some embodiments, the method further comprises a step of enriching the sample with the isolated microorganism.
In some embodiments, the method further comprises a step of depleting the isolated microorganism from the sample.
In some embodiments, the method further comprises a step of modulating a sample such that the amount, distribution, abundance, or any combination thereof, is modified so as to be compatible for administering to a subject in need thereof.
In some embodiments, modulating comprises increasing, elevating, or any equivalent thereof.
In some embodiments, modulating comprises decreasing, reducing, or any equivalent thereof.
In some embodiments, the modulating is tailored to the physical state of the subject.
In some embodiments, the method further comprises a step of determining the condition of the subject prior to administration.
In one embodiment, wherein a subject is determined to comprise a pathogenic microorganism, the method comprises depleting the pathogenic microorganism from a sample or a fraction thereof, prior to administration to the subject.
In one embodiment, wherein a subject is determined to lack a probiotic microorganism, the method comprises enriching a sample or a fraction thereof with the probiotic microorganism, prior to administration to the subject.
In some embodiments, the sample or fraction thereof is an autologous sample.
In some embodiments, the sample or fraction thereof is an allogeneic sample.
In some embodiments, the method further comprises a step of determining the produced antibody has increased specific affinity to the one selected target microorganism compared to control.
In some embodiments, the composition is for use in treatment of a subject afflicted with a disease.
In some embodiments, the disease is selected from the group consisting of: Clostridium difficile diarrhea, inflammatory bowel disease, irritable bowel disease, cancer, and diabetes.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
In some embodiments, a method for isolating a target microorganism from a sample comprising a plurality of microorganisms, is provided.
In some embodiments, the method comprises contacting a sample with an antibody having specific affinity to the target microorganism, wherein the antibody is produced by immunizing or immunization of a host organism using a selected target microorganism, wherein the selected target microorganism is selected by contacting a fraction of the sample with one or more polynucleotide molecules each having specific affinity to one target microorganism, and determining the presence of the target microorganism in the fraction of the sample, thereby isolating a target microorganism from the sample.
In some embodiments, contacting is under conditions sufficient to enable the binding of an antibody to an antigen or an epitope thereof, e.g., such as the antibody was raised against by immunizing a host organism.
In some embodiments, contacting is under conditions sufficient to enable base pairing of the one or more polynucleotides and complementary polynucleotides comprised by the one or more microorganisms of the sample.
In some embodiments, the method comprises a step of providing a sample comprising a plurality of microorganisms. As used herein, the term “providing a sample” comprises obtaining or producing the sample.
In some embodiments, the method comprises a step of contacting a fraction of the sample with one or more polynucleotide molecules each having specific affinity to one target microorganism, and determining the presence of the one or more target microorganism in the sample.
In some embodiments, determining comprises detecting a signal indicative of the hybridization of the one or more polynucleotides having specific affinity to one target microorganism. In some embodiments, hybridization comprises base pairing of the one or more polynucleotides and complementary polynucleotides comprised by the one or more microorganisms of the sample. In some embodiments, the complementary polynucleotides comprised by the one or more microorganisms of the sample comprises DNA and/or RNA polynucleotides.
In some embodiments, the signal indicative of the hybridization comprises any one of: a fluorescent signal, a radioactive signal, and a chromatic signal.
In some embodiments, the one or more polynucleotides having specific affinity to one target microorganism is any one of: fluorescently labeled, radioactively labeled, and chromatically labeled. In some embodiments, the one or more polynucleotides having specific affinity to one target microorganism comprises a molecule or a moiety embedded or incorporated therein. In some embodiments, the molecule or moiety are further recognized and/or bound by a molecule having increased binding affinity to the molecule or moiety, such as a specific antibody (e.g., digoxigenin (DIG) and an anti-DIG antibody) or a binding counterpart (e.g., avidin and biotin). In some embodiments, the antibody or binding counterpart is further linked to an enzyme. In some embodiments, the linked enzyme is capable of catalyzing colorimetric reaction. In some embodiments, the colorimetric reaction comprises a bioluminescent reaction or a chemiluminescent reaction.
In some embodiments, the method comprises a step of selecting the one or more target microorganisms determined to be present in the sample and immunizing a host organism using one of the selected target microorganisms, thereby producing an antibody having specific affinity to the one selected target microorganism.
Methods for immunization of a host organism are common and would be apparent to one of ordinary skill in the art. Non-limiting examples of a host organism suitable for immunization include, but are not limited to, a chicken, a rabbit, and a mouse.
In some embodiments, the method comprises a step of contacting the sample with the produced antibody having specific affinity to the one selected target microorganism, thereby isolating a target microorganism from the sample.
In some embodiments, the method comprises contacting the sample with a plurality of polynucleotide molecules each having specific affinity to one target microorganism.
According to some embodiments, there is provided a method for selecting one or more target microorganisms for immunizing a host organism, the method comprising the steps of: (1) providing a fraction of a sample comprising a plurality of microorganisms; (2) contacting the fraction of the sample with one or more polynucleotide molecules each having specific affinity to one target microorganism, and determining the presence of the one or more target microorganism in the fraction of the sample; and (3) selecting the one or more target microorganisms determined to be present in the fraction of the sample for immunizing a host organism.
In some embodiments, the method further comprises immunizing a host organism using one of the selected target microorganisms determined to be present in the sample or a fraction thereof. In some embodiments, the produced antibody is characterized by having specific affinity to the selected target microorganism.
In some embodiments, the method further comprises a step comprising contacting a sample with the produced antibody. In some embodiments, the method comprises a step of contacting a sample with the produced antibody having specific affinity to the selected target microorganism. In some embodiments, the contacting comprises or results in isolating the target microorganism from the sample or a fraction thereof.
As used herein, the term “plurality” refers to any number or value that is greater than one. In some embodiments, a plurality comprises 2 to 10, 2 to 50, 20 to 100, 2 to 500, 2 to 1,000, or 2 to 10,000. Each possibility represents a separate embodiment of the invention. In some embodiments, a plurality comprises at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 10,000, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
In some embodiments, the microorganism is selected from: bacteria, fungi, archaea, protozoa, and algae.
In some embodiments, the bacteria comprises a Bifidobacterium. In some embodiments, Bifidobacterium comprises B. fragilis.
In some embodiments, the targeted microorganism is a pathogenic microorganism. As used herein, the term “pathogenic microorganism” comprises any microorganism which diverts a host organism from homeostasis. In some embodiments, a pathogenic microorganism induces, initiates, promotes, propagates, or any equivalent thereof, of a disease or a symptom associated therewith.
In some embodiments, the targeted microorganism is a probiotic microorganism. As used herein, the term “probiotic microorganism” refers to any microorganism which has or promotes health beneficiary effects on a host organism comprising thereof.
In some embodiments, the selected microorganism is a specific species or a specific strain of a microorganism.
In some embodiments, the sample is selected from: a sample derived from a subject, a soil sample, and a water sample.
In some embodiments, the sample comprises environmental sample. In some embodiments, the method of the invention is directed to isolation of uncultured microorganisms.
In some embodiments, a sample derived from a subject comprises a tissue or a cell of the subject. In some embodiments, a sample derived from a subject comprises bodily fluids of the subject. In some embodiments, a sample derived from a subject comprises a stool sample of the subject.
In some embodiments, the method further comprises a step of enriching a sample with the isolated microorganism.
In some embodiments, the method further comprises a step of depleting the isolated microorganism from the sample. In some embodiments, depleting comprises: removing, eliminating, omitting, or any equivalent thereof, as long as isolated microorganism is not present in the sample. In some embodiments, the sample is devoid of the isolated microorganism upon performing the method of the invention.
In some embodiments, the method, further comprises a step of determining the produced antibody has increased specific affinity to the one selected target microorganism compared to control.
In some embodiments, the method of the invention provides a composition suitable for any application selected from: human-related, marine-related, agriculture, uncultured bacteria, health-relevant bacteria, depletion of bacteria from stool transplantation, and directed bacteria for probiotics.
As used herein, control comprises a microorganism which is not present in the sample before preforming the method of the invention (“pre-treated sample”). In some embodiments, the control is any compound and/or microorganism which does not cross react with the produced antibody. In some embodiments, the control is a microorganism of any different species, line, strain, Glade, phyla, or any equivalent thereof, other than the target microorganism.
As used herein, increased is by at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, at least 750%, at least 1,000%, or any value and range therebetween, compared to control. Each possibility represents a separate embodiment of the invention. In some embodiments, increased is by 5-150%, 10-250%, 5-400%, 10-550%, 50-600%, 100-375%, 250-750%, 300-800%, or 725-1,000%, compared to control. Each possibility represents a separate embodiment of the invention.
Methods for determining antibody specificity are common and would be apparent to one of ordinary skill in the art. Non-limiting examples of such methods, include, but are not limited to, western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA, including direct and indirect ELISA), competitive binding assays, and others.
In some embodiments, the present invention provides a sample obtained by the method of the invention.
In some embodiments, the present invention provides a composition comprising a sample obtained by the method of the invention.
In some embodiments, the composition further comprises an acceptable carrier. In some embodiments, the carrier comprises a pharmaceutically acceptable carrier.
In some embodiments, the composition is for use in treating a subject afflicted with a disease.
In some embodiments, the disease is selected from: Clostridium difficile diarrhea, inflammatory bowel disease (IBD), irritable bowel disease, cancer, and diabetes.
In some embodiments, the composition is suitable for use in cancer immunotherapy.
In some embodiments, the composition is a functional probiotic composition. In some embodiments, the functional probiotics is suitable for use in treating an infant. In some embodiments, the functional probiotic composition comprises Bifidobacterium.
As used herein, the term “cancer” refers to a disease associated or characterized by abnormal cell proliferation. In some embodiments, cancer refers to a disease comprising cell proliferation.
In some embodiments, IBD comprises Crohn's disease, ulcerative colitis, or both.
The terms “sample” and “fraction of a sample” are used herein interchangeably.
In some embodiments, the present invention is directed to a method for producing an antibody, comprising: (a) contacting a sample with one or more polynucleotide molecules each having specific affinity to one target microorganism; (b) selecting a target microorganism determined to be present in the sample; and (c) immunizing a host organism using the selected target microorganism, thereby producing the antibody.
In some embodiments, a composition comprising the produced antibody is provided.
In some embodiments, the produced antibody, a composition comprising thereof, or both, is for use in isolating a microorganism from a sample comprising a plurality of microorganisms.
In some embodiments, the antibody is a chordate derived antibody. In some embodiments, the antibody is a non-mammalian antibody. In some embodiments, the antibody is an avian-derived antibody. In some embodiments, the antibody is a shark derived antibody. In some embodiments, the antibody is a chicken derived antibody. In some embodiments, the antibody is an immunoglobulin Y (IgY) antibody. In some embodiments, has no or low cross-reactivity with a microorganism cell wall or a component thereof. In some embodiments, the antibody has no or low cross-reactivity with lipopolysaccharide.
Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6th Edition) by Leroy “Skip” G Wade; Organic Chemistry by T. W. Graham Solomons and, Craig Fryhle.
FISH probes were designed to a bacterial specific unique complementary 16S ribosomal RNA (rRNA) gene sequence, at the level of DNA. Fluorophore-labeled probe was ordered from Biomers (Germany). Up to 1×109 of cultured bacteria or stool washed bacteria were fixed in 50% ice cold ethanol at −20° C. for 20 minutes. Following washing steps with hybridization buffer (0.9 M NaCl, 20 mM Tris pH 7.5, 0.01% SDS, 20% HiDi Formamide), bacteria were resuspended in 50 μl of hybridization buffer and probes (2 pmole/μl). Following 2 hours of incubation at 46° C. bacteria were washed with washing buffer (215 mM NaCl, 20 mM Tris pH 7.5, 5 mM EDTA) at 48° C. for 15 minutes 3 times.
For generating bacteria specific antibody, laying chickens were vaccinated subcutaneous with 50 μg of isolated bacteria, emulsified in Freund incomplete adjuvant (Sigma). Two weeks after a second boost injection, IgY antibodies were collected from the egg yolks, followed by sodium sulfate purification step. Antibody specificity was tested by flow cytometry.
Bacteria were washed with FACS buffer (PBS, 2% FCS, 1 mM EDTA, and 0.1% Sodium azide) and re-suspended in a concentration equivalent to 100 events/second (Fortessa low speed).
Washed bacteria were incubated with the purified antibody for 30 minutes at 4° C., followed by a secondary goat anti-chicken IgY Alexa Fluor-647 conjugated.
Magnetic beads (Thermo-Scientific) were coated with mouse anti-B. fragilis antibody. Beads were incubated with 1:1,000 Hoechst stained-bacteria for 30 minutes at 4° C. and analyzed by flow cytometry.
Bacteria are stained with 1:2,000 B. fragilis specific antibodies (Secondary Ab— 1:2,000 Rabbit (Fab2) anti Chicken IgY APC conjugate). Staining works at 4° C. and 25° C. in established staining buffer. Washing steps-1 ml staining buffer, centrifuge 6,500 g for 5 minutes. Optimal thresholds and voltage values were set on the AriaIII BD FACS. As control, the following groups were used: (1) Single stained bacteria; (2) Each bacteria with B. fragilis; and (3) Mix of all. In the mixtures, bacteria mixed, and then stained with specific Ab.
Staining and Isolating B. fragilis from Feces
B. fragilis were stained by metabolic click labeling— AF488 fluorophore. Fecal bacteria were washed and mixed with labeled B. fragilis and were detected by specific antibodies— APC fluorophore.
Briefly, bacteria were stained with 1:2,000 B. fragilis specific antibodies (Secondary Ab— 1:2,000 Rabbit (Fab2) anti Chicken IgY APC conjugate). Staining was performed at 25° C. in staining buffer (PBS, 10% FBS, 0.5 mM EDTA, pH 7.2). Washing steps-1 ml staining buffer, centrifuge 6,500 g for 5 minutes at 4° C. Optimal thresholds and voltage values were set for LSR Fortessa, BD analyzer.
Sorting B. fragilis from Feces
B. fragilis were stained for nucleic acids using Hoechst— AF405 Channel. Fecal bacteria were washed and mixed with Hoechst stained B. fragilis and were detected by specific antibodies— APC fluorophore.
Briefly, bacteria were stained with 1:2,000 B. fragilis specific antibodies (Secondary Ab— 1:2,000 Rabbit (Fab2) anti Chicken IgY Alexa Fluor 647 conjugate). Staining was performed at 25° C. in staining buffer (PBS, 10% FBS, 0.5 mM EDTA, pH 7.2). Washing steps-1 ml staining buffer, centrifuge 6,500 g for 5 minutes at 4° C. Optimal thresholds and voltage values were set for AriaIII, BD sorter. Bacteria were sorted to 4 tubes and were analyzed again by re-running the sorted bacteria.
Production of Specific Antibodies Raised Against FISH-Isolated Bacteria
The inventors have shown that bacteria specific detection is feasible using complementary fluorescence labeled probes (
To verify the ability of the antibody-coated magnetic beads to specifically bind bacteria, beads were incubated with Hoechst stained B. fragilis and tested by flow cytometry (
Detecting and Sorting Bacteroides fragilis in Feces
The inventors showed that cultured bacteria were recognizable with high specificity and efficiency (
While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/969,197 titled: “METHOD FOR ISOLATING A MICROORGANISM”, filed Feb. 3, 2020, the contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2021/050128 | 2/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62969197 | Feb 2020 | US |
