Patent Grant
11299520
Clostridium difficile infection (CDI) is the leading cause of antibiotic- and healthcare-associated diarrhea and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics and outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few.
CDI affects 500,000-1,000,000 patients annually in the USA, and morbidity and mortality due to this pathogen have increased over the past 15 years. Currently, the only FDA-approved treatment involves prolonged antibiotic use, which is ironically, associated with disease relapse and the threat of burgeoning antibiotic resistance. CDI is precipitated when commensal flora are suppressed following antibiotic treatment. The use of antibiotics can suppress the protective normal microbiota causing susceptibility to infection. Exposure to C. difficile spores results in colonization of the host gastrointestinal tract. Current treatments include use of antibiotics, which alter the bacterial composition of the gut microbiome. Vaccines are being developed for preventing C. difficile disease, but do not protect against C. difficile colonization.
Bacterial adherence is thought to be an important C. difficile virulence attribute, with surface-layer proteins (SLPs) playing key roles (Merrigan et al., PLoS ONE 8, e78404 (2013)). C. difficile elaborates up to 29 different SLPs, which are displayed in para-crystalline architecture on the cell surface. C. difficile SLPs are also implicated in immune modulation; thus, they are critical non-toxin virulence factors. While SLPs have been proposed as anti-CDI vaccine candidates, the SLP-based vaccine trials have not been successful most likely due to the variability in SLP epitope antigenicity (Biazzo et al., J Med Microbiol 62, 1444-1452 (2013)). At present, effective treatments and preventatives for CDI and colonization are lacking.
There is an urgent need in the art for new compositions and methods for CDI treatment. This invention addresses this need.
In one aspect, the invention includes a chimeric nucleic acid molecule comprising a nucleic acid sequence having a phosphoglycerate mutase (pgm) constitutive promoter (pgm promoter) and a nucleic acid sequence encoding a chimeric bacterial surface layer protein A (SlpA) comprising a Clostridium difficile derived host-cell binding domain and a lactic acid bacterium (LAB) derived peptidoglycan anchor.
In another aspect, the invention includes a chimeric polypeptide encoded by a nucleic acid sequence comprising a phosphoglycerate mutase (pgm) constitutive promoter (pgm promoter) and a nucleic acid sequence encoding a bacterial surface layer protein A (SlpA) comprising a Clostridium difficile derived host-cell binding domain and a lactic acid bacterium (LAB) derived peptidoglycan anchor.
In yet another aspect, the invention includes a vector comprising a nucleic acid sequence comprising a chimeric surface layer protein A (slpA) gene of Lactobacillus acidophilus and Clostridium difficile; a pgm promoter; an ytvA fluorescent allele from B. subtilis; a temperature sensitive repA gene; a chloramphenicol resistance cat gene; an internal fragment of Lactobacillus acidophilus thymidylate synthase A (thyA); and an internal fragment of Lactobacillus casei thyA.
In yet another aspect, the invention includes an engineered cell comprising the aforementioned chimeric nucleic acid molecule and the aforementioned vector.
In yet another aspect, there is provided a method of treating or preventing Clostridium difficile infection in a mammal. The method comprises administering to the mammal the engineered cell, thereby treating or preventing Clostridium difficile infection in the mammal.
In yet another aspect, the invention is a method of colonizing the gut of a mammal with an engineered lactic acid bacterium (LAB). The method comprising administering to the mammal the engineered cell, thereby colonizing the gut of the mammal with the LAB
In certain embodiments, the pgm of the chimeric nucleic acid molecule is from a Lactobacillus species selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In certain embodiments, the LAB of the chimeric nucleic acid molecule is from a Lactobacillus species selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In certain other embodiments, the nucleic acid sequence comprising the pgm promoter and the nucleic acid sequence encoding the SlpA has the nucleic acid sequence of SEQ ID NO: 18.
In certain embodiments, the pgm of the chimeric polypeptide is from a Lactobacillus species selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In certain embodiments, the LAB of the chimeric polypeptide is selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In certain other embodiments, the nucleic acid sequence comprising the pgm promoter and the nucleic acid sequence encoding the SlpA have the nucleic acid sequence of SEQ ID NO: 18.
In certain embodiments, the pgm of the vector is from a Lactobacillus species selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In certain embodiments, the vector is a Lactobacillus expression vector. In certain embodiments, the vector is pMGM15. In certain embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 1.
In certain embodiments, the chimeric nucleic acid molecule or the nucleic acid sequence encoding the vector is integrated into the chromosome of the engineered cell. In certain embodiments, the engineered cell comprises a LAB selected from the group consisting of a human-derived Lactobacillus species, a human-derived Lactobacillus acidophilus, and a human-derived Lactobacillus casei.
In certain embodiments, the administered engineered cell is selected from the group consisting of a human-derived Lactobacillus, a human-derived Lactobacillus acidophilus, and a human-derived Lactobacillus casei. In certain embodiments, the engineered cell is administered orally. In certain other embodiments, the engineered cell is administered daily. In certain embodiments, the mammal is a human.
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “Surface-Layer Protein A (SlpA)” is meant a polypeptide or fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided at NCBI Accession No. CAJ69681 or WP_011254065 and having bacterial adherence activity. Exemplary SlpA amino acid sequences are provided below:
Clostridium difficile SlpA (full length sequence)
Lactobacillus acidophilus SlpA ortholog cell-wall anchor
By “SlpA nucleic acid molecule” is meant a polynucleotide encoding SlpA.
By “SlpA variable domain” is meant a polypeptide having 85% identity to the following sequence (SEQ ID NO: 15):
In one embodiment, the SlpA variable domain is from C. difficile SlpA.
By “SlpA cell wall binding domain” is meant a polypeptide having 85% identity to the following sequence (SEQ ID NO: 16):
In one embodiment, the SlpA cell wall binding domain is from Lactobacillus (e.g., Lactobacillus acidophilus).
By “chimeric SlpA” is meant a polypeptide having two or more SlpA sequences from two or more bacterial strains. In one embodiment, a chimeric SlpA has an SlpA variable domain from C. difficile SlpA and an SlpA cell wall binding from Lactobacillus acidophilus.
In another embodiment, a chimeric SlpA has an SlpA signal sequence from Lactobacillus acidophilus, an SlpA variable domain from C. difficile SlpA and an SlpA cell wall binding from Lactobacillus acidophilus. An exemplary chimeric SlpA sequence is provided below (SEQ ID NO: 17):
By “undesirable gut microbiome” is meant a community of microbes comprising a pathogen or having a biological activity associated with a pathogenic process. In one embodiment, an undesirable gut microbiome comprises an increased number or percentage of Clostridium difficile relative to the number or percentage of C. difficile present in the gut of a healthy control subject.
By “normal gut flora” is meant a population of microbes that is substantially similar to the population of microbes present in the gut of a healthy control subject.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
By “effective amount” is meant the amount of a composition of the invention (e.g., comprising a probiotic bacteria expressing Clostridium difficile SlpA, or fragment thereof) required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of probiotic bacteria of the invention varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “expression vector” is meant a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as plasmids, cosmids, and viruses that incorporate the recombinant polynucleotide.
A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference polypeptide or nucleic acid molecule. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids or nucleotides.
The term “human-derived bacteria” means bacterial species that have been isolated from a fecal sample or from a gastrointestinal biopsy obtained from a human individual or whose ancestors were isolated from a fecal sample or from a gastrointestinal biopsy obtained from a human (e.g., are progeny of bacteria obtained from a fecal sample or a gastrointestinal biopsy). For example, the bacterial species may have been previously isolated from a fecal sample or from a gastrointestinal biopsy obtained from a human and cultured for a sufficient time to generate progeny. The progeny can then be further cultured or frozen. The human-derived bacteria are naturally occurring commensals that populate the gastrointestinal tract of human individuals, preferably healthy human individuals.
The terms “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. Thus, a purified isolated bacterium that is part of a culture or inoculum is at least about 90%, 95% or 100% free of bacteria, fungi, viruses, or other undefined microbes.
By “marker” is meant any analyte associated with a disease or disorder.
The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
“Pharmaceutically acceptable” refers to those properties and/or substances that are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
As used herein, the term “pharmaceutical composition” or “pharmaceutically acceptable composition” refers to a mixture of at least one compound or molecule useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound or molecule to a patient. Multiple techniques of administering a compound or molecule exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, rectal and topical administration.
As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound or molecule useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound or molecule useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.
By “reference” is meant a standard or control condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
The invention provides a probiotic human-derived lactic acid bacteria (LAB) (e.g., Lactobacillus) expressing a chimeric nucleic acid molecule, and its use for colonizing the gut or digestive tract of a subject to treat CDI. The invention also provides a method of treating or preventing Clostridium difficile infection and colonization. The invention features use of the probiotic bacteria of the invention for the replacement of a gut microbiome associated with disease.
The invention is based, at least in part, on the discovery that expression of a chimeric SlpA from Clostridium difficile and Lactobacillus acidophilus, in an engineered LAB (e.g. Lactobacillus acidophilus) is effective for colonizing the gut with the engineered LAB. It was also found that the engineered LAB of the invention protected the gut from virulent Clostridium difficile challenge. These findings indicate that administration of an engineered LAB expressing a chimeric SlpA from Clostridium difficile and Lactobacillus acidophilus is useful for treating or preventing Clostridium difficile infection and colonization.
Compositions
Surface-Layer Protein a (SlpA) and Expression Thereof
Many gram-positive bacteria including C. difficile possess a surface-layer that covers the peptidoglycan-rich cell wall. The most abundant Surface layer protein in C. difficile is slpA, a major contributor of adhesion to, and colonization of, intestinal epithelial cells. (Merrigan et al. PLoS ONE 8(11): e78404,2013). SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.
In one aspect, the invention provides a chimeric nucleic acid molecule comprising a nucleic acid sequence comprising a phosphoglycerate mutase (pgm) strong constitutive promoter (pgm promoter) and a nucleic acid sequence encoding a bacterial surface layer protein A (SlpA) consisting of a Clostridium difficile derived host-cell binding domain and a lactic acid bacterium (LAB) derived peptidoglycan anchor. In some embodiments, the LAB is a Lactobacillus species selected from the group consisting of Lactobacillus acidophilus or Lactobacillus casei. In some embodiments, the nucleic acid sequence comprising the pgm promoter and the nucleic acid sequence encoding the chimeric SplA gene from Clostridium difficile and Lactobacillus acidophilus has the following sequence (SEQ ID NO: 18):
In another aspect, the invention provides a chimeric polypeptide encoded and expressed by the nucleic acid sequence described above herein.
Expression of a Chimeric Polypeptide
In order to express the chimeric polypeptide of the invention, DNA molecules obtained by any of the methods described herein or those that are known in the art, can be inserted into appropriate expression vectors by techniques well known in the art. For example, a double stranded DNA can be cloned into a suitable vector by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation. DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.
Therefore, the invention includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules encoding genes) as described herein. In some embodiments, the vector of the invention is a recombinant vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived. For example, a recombinant vector may include a nucleotide sequence encoding an SlpA chimeric polypeptide operatively linked to regulatory sequences, e.g., promoter sequences, terminator sequences, and the like, as defined herein. Recombinant vectors which allow for expression of the genes or nucleic acids included in them are referred to as “expression vectors.”
In one aspect, although the pgm promoter is exemplified herein, the invention should not be construed to be limited solely to this promoter sequence. In certain embodiments, promoter sequences that are useful in the invention include any promoter known in the art that induces high levels of expression of the gene of interest.
The vector may also include conventional control elements which are operably linked to a gene of interest in the vector (e.g. a heterologous gene) in a manner which permits its transcription, translation and/or expression in a cell infected with the vector produced by the invention (i.e. pMGM15).
As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. There are numerous expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art that may be used in the compositions of the invention.
Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements may function either cooperatively or independently to activate transcription.
In one embodiment, a suitable promoter is the pgm promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other bacterial constitutive promoter sequences may also be used, including, but not limited to the T7 promoter and Sp6 promoter. Further, the invention should not be construed to be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a Lac promoter, an araBAD promoter, and a pL promoter.
In order to assess the expression of the heterologous gene of interest (e.g. chimeric SlpA), the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transformed through the vector (e.g. pMGM15). In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a transformation/protoplating/electroporation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as the chloramphenicol resistant gene and the like.
Reporter genes are used for identifying potentially infected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene such as ytvA from B. subtilis (Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
In some of the molecules of the invention described herein, one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the invention are operatively linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a prokaryotic host cell. Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector. A selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use.
Factors of importance in selecting a particular vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
Once the vector(s) is constructed to include a DNA sequence for expression, it may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
After the introduction of one or more vector(s), host cells (e.g. Lactobacillus sp.) are usually grown in a selective medium, which selects for the growth of vector-containing cells. Expression of recombinant proteins can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence. Purification of recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any conventional procedures involving extraction, precipitation, chromatography and electrophoresis. A further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein. Generally, crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.
In one aspect, the invention provides a vector comprising a nucleic acid sequence comprising: (a) a chimeric surface layer protein A (slpA) gene of Lactobacillus. acidophilus and Clostridium difficile; (b) a phosphoglycerate mutase (pgm) constitutive promoter; (c) an ytvA fluorescent allele from B. subtilis; (d) a temperature sensitive repA gene; (e) a chloramphenicol resistance cat gene; (0 an internal fragment of Lactobacillus acidophilus thymidylate synthase A (thyA); and, (g) an internal fragment of Lactobacillus casei thyA.
In some embodiments, the vector is a Lactobacillus expression vector. In some embodiments, the vector is pMGM15. In other embodiments, the vector has a nucleic acid sequence of SEQ ID NO: 1 (as detailed in
In one aspect the invention provides an engineered cell comprising the chimeric nucleic acid molecule described above herein.
In one aspect the invention provides an engineered cell comprising the vector described above herein.
In some embodiments, the chimeric nucleic acid molecule or the nucleic acid sequence encoding the vector is integrated into the chromosome of the engineered cell. In some embodiments, the engineered cell is a human-derived Lactobacillus, a human-derived Lactobacillus acidophilus, or a human-derived or milk-derived Lactobacillus casei.
Therapeutic Compositions
The invention features engineered probiotic bacteria expressing Clostridium difficile SlpA or fragment thereof (e.g., chimeric SlpA). In particular, Clostridium difficile SlpA, or fragment thereof, is expressed in lactic acid bacteria (LAB) particularly Lactobacillus cells (e.g., Lactobacillus acidophilus or Lactobacillus casei). In additional embodiments, one or more strains of probiotic bacteria expressing a chimeric SlpA polypeptide are administered or formulated as a therapeutic composition. In certain embodiments, the SlpA expressed is a chimeric SlpA comprising a C. difficile SlpA variable domain and Lactobacillus (e.g., Lactobacillus acidophilus or Lactobacillus casei) SlpA cell wall binding domain. The SlpA additionally includes a bacterial secretion signal that is appropriate for surface expression in its host cell (e.g., Lactoccocus, Lactobacillus, Lactobacillus acidophilus or Lactobacillus casei). In various embodiments, the invention also includes nucleic acid molecules and vectors encoding a chimeric SlpA polypeptide. Vectors encoding the chimeric SlpA polypeptide can be used to direct or regulate the expression of the chimeric SlpA by the cell (see e.g., Duong et al., Microbial Biotechnology 2010, 4(3): 357-367 which is herein incorporated in its entirety by reference). Such vectors contain one or more origin of replication sequences that can be used by a bacterial cell, promoter sequences for expression in a bacterial cell (e.g., constitutive or inducible), genetic markers for selection (e.g., antibiotic resistance); origin of transfer sequences for bacterial conjugation (e.g., traJ), and may also be codon optimized for protein expression.
Alternatively, nucleic acid sequences encoding chimeric SlpA may be integrated into the Lactobacillus genome. In certain embodiments, nucleic acid sequences encoding the chimeric SlpA can be integrated into the Lactobacillus genome through recombination between vectors comprising the nucleic acid sequence encoding the chimeric SlpA poly peptide and bacterial chromosome. Such techniques are well known in the art (see e.g., Leenhouts, et al., Appl. Environ. Microbiol. 1989, 55(2): 394-400, and Gaspar, et al., Appl. Environ. Microbiol. 2004, 70(3): 1466-74 which are herein incorporated in their entirety by reference).
Probiotic strains may also be engineered with auxotrophic selection, for example requiring thiamine or thymine supplementation for survival.
The present invention provides methods of treating diseases or symptoms thereof associated with the presence of one or more undesirable bacteria in the gut of a subject (e.g. C. difficile). Accordingly, the invention provides compositions and methods for treating a subject having or at risk of developing a disease associated with undesirable changes in the gut microbiome, the method involving administering a therapeutically effective amount of a composition comprising a engineered probiotic lactic acid bacterium (LAB) of the invention to a subject (e.g., a mammal, such as a human). In particular, the compositions and methods of the invention are effective for treating or preventing Clostridium difficile infection, colonization, or diseases and symptoms thereof (e.g., diarrhea). Without wishing to be bound by theory, an engineered Lactobacillus expressing the chimeric Clostridium difficile/Lactobacillus acidophilus SlpA of the invention can colonize the gut and compete with Clostridium difficile for binding and colonization. Accordingly, the method includes the step of administering to a mammal a therapeutic amount of an amount of a composition comprising one or more engineered cell (i.e. LAB) the invention sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
In one aspect the invention provides a method of treating or preventing Clostridium difficile infection in a mammal. The method comprises administering an engineered LAB expressing the vector as described above herein, thereby treating or preventing Clostridium difficile infection in the mammal.
In yet another aspect the invention provides, a method of colonizing the gut of a mammal with an engineered LAB. The method comprises administering an engineered LAB expressing the vector as described above herein, thereby colonizing the gut of the mammal with the engineered LAB.
In some embodiments, the administered engineered LAB is selected from the group consisting of a human-derived Lactobacillus, a human-derived Lactobacillus acidophilus, and a human-derived Lactobacillus casei.
Identifying a subject in need of treatment for a disease associated with the gut microbiome can be conducted by a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). In certain embodiments, the subject has undergone or is undergoing treatment with antibiotics. As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of a composition comprising the probiotic bacteria of the invention to a subject (e.g., human) in need thereof. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider. The compositions herein may be also used in the treatment of any other disorders in which a microbial imbalance in the digestive tract may be implicated.
Methods of Delivery
One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route.
Routes of administration of any of the compositions of the invention include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
In one embodiment, the compositions comprising the engineered cell (LAB) of the invention may be administered orally, rectally, or enterally. Preferably the compositions are administered to a subject in tablet form, by feeding tube, by enema, or by colonoscopy. Preferably, the probiotic bacteria of the invention are diluted in a suitable excipient (e.g., saline solution). A non-limiting example of an effective dose may include 106-109 colony forming units of bacteria per day). In some embodiments, the engineered cell of the invention is administered orally. In some embodiments, the engineered cell is administered daily as needed.
Kits
The invention provides kits for colonizing probiotic engineered LAB of the invention in the gut of a mammalian host (such as a human). The invention also provides kits for the treatment or prevention of Clostridium difficile infection or colonization. In particular embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition comprising the engineered LAB of the invention; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
The kit preferably contains instructions that generally include information about the use of the composition for the expansion of the microbial consortia in the gut of the subject. The kit further contains precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
The Materials and Methods used in the performance of the experiments disclosed herein are now described.
Cell lines. The human intestinal epithelial cell line C2BBe, a brush border-expressing Caco-2 sub-clone (Peterson et al (1992), J Cell Sci, 102 (Pt 3), 581-600), was used in this study and cultured as previously reported (Roxas et al., 2014, Am J Physiol Gastrointest Liver Physiol 307(3), G374-380).
Bacterial Strains and plasmids. All strains and plasmid are described in Table 1. Lactic acid bacteria were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Specifically, Lactobacillus casei strain 334 [Orla-Jensen; (Dellaglio et al., 2002, Int J Syst Evol Microbiol, 52(Pt 1), 285-287), and Lactobacillus acidophilus strain 4356 (Roussel et al., 1993, Appl Bacteriol 74(5), 549-556) were used for these studies. Lactic acid bacteria were grown in De Man, Rogosa and Sharpe (MRS) broth (Duong et al., 2011, Microb Biotechnol, 4(3), 357-367) and incubated at 30° C. in the presence of 5% CO2. Bacteria were cultured for 3-5 days to reach saturation [>1.0×108 colony forming units (CFU) per mL].
L. acidophilus and L. casei strains ferment the dextrose in MRS to distinct products, and the corresponding pH changes can be detected by including bromophenol blue into the media (MRS-BPB)(Lee et. al., 2008, Lett Appl Microbiol 46(6), 676-681). L acidophilus, a homo-fermenter, metabolizes dextrose to lactic acid, and the plates remain violet/blue (pH>4.6); L. casei, a hetero-fermenter converts dextrose to acetic acid, and the drop in pH (<3.0) results in a color change to yellow/white. Further, L. casei, unlike L. acidophilus, can ferment mannitol, and this can be verified by growth on Purple Broth Base (Difco™ Becton, Dickinson and Company Sparks, MD); the acidic change resulting from mannitol fermentation causes the pH indicator bromocresol purple to turn yellow.
The slpA C. difficile/L. acidophilus“chimera” fragment was designed with a strong lactic-acid-bacterial (LAB) promoter [endogenous to the phosphoglycerate mutase (pgm) gene in plasmid pTRK848 (Duong et al., 2011, Microb Biotechnol, 4(3), 357-367), a lactic-acid-bacterial Shine-Dalgarno (ribosome binding site) sequence (Duong et al., 2011, Microb Biotechnol, 4(3), 357-367), a signal sequence from a Lactobacillus acidophilus S-layer protein, a codon-optimized C. difficile strain 630 host-cell-binding fragment, and the L. acidophilus SlpA-ortholog cell-wall-binding domain (Michon et al., 2016, Microb Cell Fact 15, 70). The entire fragment (F1) was chemically synthesized (DNA 2.0, now ATUM, Newark, Calif.), and cloned into the DNA 2.0 maintenance vector pJ241 to yield pMGM13. A second DNA fragment (F2) comprising a broad host-range temperature-sensitive origin of replication (repA), and a chloramphenicol resistance gene (catP) was also chemically synthesized based on sequence information obtained from the broad host-range plasmid [pKS1(Steen et al., 2010, Nature 463(7280), 559-562)]. This fragment was self-ligated to form pMGM11 (“empty vector”). The two synthesized fragments F1 and F2 harbored PmeI and FseI restriction sites at their termini; following digestion with those enzymes, linear F1 and F2 were ligated using T4 DNA ligase (Sigma, St. Louis Mo.) to generate pMGM14. Plasmid integrity of pMGM14 was confirmed by complete DNA sequencing. All plasmids were transformed into E. coli strains DH10b, TOP10 or NCK1753 for maintenance or propagation purposes, or into E. coli GCS prior to extraction for electroporation. All plasmids were PCR-verified prior to any use in lactic acid bacteria.
Lactic acid bacterial (LAB) transformation. The pMGM14 plasmid described above was first extracted from the E. coli storage strain, and 10 μg used for each LAB transformation. Lactobacillus acidophilus strain 4356 and Lactobacillus casei 343 were propagated in Mann-Rogosa-Sharpe (MRS) medium (Remel, Lenexa, Kans.) per the method of Walker et al. (Walker et al., 1996, FEMS Microbiol Lett 138(2-3), 233-237) and Kim et al. (Kim et al., 2005, Anaerobe 51, 42-46). In brief, LAB strains were grown to saturation at 37° C. in 5% CO2, sub-cultured at a 1:50 dilution, and re-grown using the same process two more times. To shear cell-wall proteins, and prepare the resulting protoplasts for electroporation, a fourth sub-culture was grown for 15 hours in MRS supplemented with 1% glycine, and then sub-cultured in the same medium at a 1:50 dilution for an additional 6 hours. Bacteria were harvested using centrifugation, and chilled pellets subjected to electroporation with 10 μg plasmid DNA, followed by recovery in MRS broth for 3 hours, and growth on MRS-agar+5 μg/mL chloramphenicol (Sigma, St. Louis, Mo.) at 30° C. in 5% CO2. Plasmid was extracted from one-half of a colony of multiple purified transformants, and verified via DNA sequence analysis. The remaining bacterial colonies were propagated in the presence of chloramphenicol at 42° C., the non-permissive temperature, at which repA is non-functional, thus selecting for integrants. For pMGM14-based transformants, putative integrants were purified, and assessed for chimeric slpA presence by PCR. All confirmations were performed at the non-permissive temperature. Three independently-isolated and PCR-verified identical transformants were bio-banked for the L. casei strain (herein referred to as Syn-LAB 2.0 clones), and seventeen independently-isolated and PCR-verified identical transformants for the L. acidophilus strain (herein referred to as Syn-LAB 2.1 clones). For in vitro studies only, all LAB strains were propagated at the non-permissive temperature with antibiotic selection as appropriate (5 μg/mL chloramphenicol). However, for all in vivo studies, while strains were propagated as above, no selection antibiotic was administered to the animals.
An identical procedure was used to generate empty-vector harboring LAB strains (Table 1); these strains were PCR-verified, and used only for in vivo studies. Plasmids were maintained episomally; the lack of homology with the LAB host limited the possibility of integration of vector sequences into the chromosome.
Transepithelial electrical resistance (TEER) measurements. Polarized human intestinal epithelial cells [C2BBe; (Peterson et al., 1992, J Cell Sci 102 (Pt 3), 581-600.)] were grown on 0.33-cm2 collagen-coated Corning™ Transwells (Thermo Fisher Scientific) for 14 days. Monolayers were treated apically with 1.42×107 cfu/well and 1.96×107 cfu/well of parental and Syn-LAB 2.0 respectively. Measurements were made every hour for 7 hours, and at 24 hours post-treatment using an epithelial volt-Ohm voltmeter (World Precision Instruments, Sarasota, Fla.), and TEER calculated by applying Ohm's Law. An identical setup was used when testing Syn-LAB 2.1 and its L. acidophilus parent strain.
Host cell survival measurement. C2BBe monolayers were treated with media alone, the parent LAB strain, or the corresponding Slp chimera-expressing Syn-LAB derivatives, and host cell viability was assessed using the propidium iodide (PI) uptake assay as described previously (Roxas et al., 2012, Infect Immun 80(11), 3850-3857). Briefly, PI (2 μg/ml; Molecular Probes) was added to the treated cells, and fluorescence measured after 30 minutes using a microplate reader (Synergy HT; BioTek instruments, Winooski, Vt.). To estimate maximal PI uptake, a set of wells were treated with 70% methanol prior to PI treatment.
Immunoblot Analyses. Surface layer proteins (Slp) were extracted from Lactobacillus parent and Syn-LAB strain saturated cultures (OD600 nm=1.5) using 0.2M glycine (pH2.2), as described by Calabi et al (Calabi et al., 2001, Mol Microbiol 40(5), 1187-1199). For dot blot analyses, 31.25 ng, 62.5 ng, 125 ng, 500 ng, 1 μg and 2 μg of total protein were blotted on nitrocellulose membranes (Bio-rad, Richmond, Calif.). Slp extracts (5-10 μg) were also separated on 4-20% TGXTM pre-cast protein gels (Bio-rad, Richmond, Calif.). Separated proteins were transferred to 0.2-pin nitrocellulose membranes (Transblot Cell Apparatus, Bio-Rad). Blots were blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST) for 1 hour, incubated with antiserum specific to C. difficile SlpA (raised against the C. difficile strain 630 SlpA (Merrigan et al., 2013, PLoS One 8(11), e78404) overnight at 4° C. and in horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 hour at room temperature (Sigma-Aldrich, St. Louis, Mo.). Membranes were washed five times for 5 minutes in blocking solution between each incubation step and developed with SuperSignal West Femto Chemiluminescent Substrate (ThermoFisher Scientific, Rockford, Ill.).
Immunofluorescence Microscopy. SlpA chimera expression in L. casei WT and Syn-LAB 2.0 and Syn-LAB 2.1 strains was evaluated via immunofluorescence staining using antiserum specific to C. difficile SlpA. Lactobacillus sp cultures were allowed to settle for 10 minutes in 12-well plates with poly-L-lysine-coated coverslips. Unattached bacteria were removed, and samples were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes, permeabilized with 0.2% Triton X-100 in PBS for 15 minutes, quenched with 50 mM NH4Cl and 0.125M glycine in PBS for 15 minutes, and blocked with 5% IgG-free bovine serum albumin (BSA) in PBS for 1 hour. Samples were incubated with antiserum specific to C. difficile SlpA overnight at 4° C., and then washed three times with 1% IgG-free BSA in PBS. Secondary antibodies (Alexa 488-conjugated goat anti-rabbit IgG antisera; Thermo Fisher Scientific, Waltham, Mass.) were added at 8 μg/ml in 5% IgG-free BSA for 1 hour. Samples were mounted in ProLong Diamond Antifade reagent (Thermo Fisher Scientific, Waltham, Mass.). Intestinal tissue samples (ileum, cecum, and colon) from LGV Golden Syrian Hamsters (Charles River Laboratories, San Diego, Calif.) treated with Syn-LAB 2.0 were frozen in OCT embedding medium (Tissue-Tek, Sakura Finetek, CA) and stored at −80° C. OCT-mounted tissue samples cut at 3 micron thickness were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes, and processed for SlpA immunofluorescence staining as described above. Samples were stained with 4,6-diamidino-2-phenylindole (DAPI) prior to mounting in ProLong Diamond Antifade reagent. Images were captured using EVOS® FL Imaging System (Thermo Fisher Scientific, Waltham, Mass.) or DeltaVision Elite Deconvolution Microscope (GE Healthcare, Pittsburgh, Pa.).
Flow cytometry. Parent and transformed Lactobacillus sp strains were cultured in MRS broth as described above, and subjected to Gram's staining to verify purity and morphology. Bacteria were pelleted by centrifugation at 4000 g for 2 minutes. Bacterial pellets were washed gently three times with blocking solution (2% IgG-free BSA in PBS) and then incubated with antiserum specific to C. difficile SlpA for 30 minutes. Secondary antibodies (Alexa Fluor 555-conjugated goat anti-rabbit IgG antisera; Thermo Fisher Scientific, Waltham, Mass.) were added at 8 μg/ml in 2% IgG-free BSA for 30 minutes. Samples were washed three times with blocking solution after each antibody incubation step. Stained samples were re-suspended in blocking solution at 106 cells/mL density and analyzed via flow cytometry using a BD FACSCANTO II machine (BD Biosciences, San Jose, Calif.). List mode data files consisting of 10,000 events gated on FSC (forward scatter) vs SSC (side scatter) were acquired and analyzed using FACSDiva 8.0.1 software (BD Biosciences, San Jose, Calif.). Appropriate electronic compensation was adjusted by acquiring the cell populations stained with the fluorophore, as well as an unstained control.
Golden Syrian hamster studies. All hamster studies were approved by the Institutional Animal Care and Use Committee of the University of Arizona. The Golden Syrian hamster model was employed to study both colonization/shedding and protection conferred by Syn-LAB strains. For all studies, male hamsters (6-8 weeks; 90-110 g weight) were used. Shedding studies: Prior to any treatment, hamster stool plated on MRS yielded no colonies, confirming that the animals were devoid of endogenous Lactobacillus bacteria. Animals received a daily dose of 108 Syn-LAB 2.0 or 108 Syn-LAB 2.1 respectively. Feeding and enumeration were continued for six days. For Syn-LAB 2.0-treated animals only, oral clindamycin (prescription solution; clindamycin sulfate; University of Arizona Pharmacy; 30 mg/kg) was administered on Day 4, and LAB detection monitored until Day 6. Fecal pellets were collected daily, and pellets were re-suspended in PBS, homogenized, serially diluted and plated on the appropriate Syn-LAB selective medium containing chloramphenicol. Colonies were detected only in stool samples from Syn-LAB-treated animals, and not from untreated controls. Shedding from all animals was statistically indistinguishable. For added confirmation, select colonies were 16S PCR-verified for L. casei as well as the presence of the chimeric slpA.
Challenge studies: These were performed in two modalities, a “fixed-dose” and a “continuous dose” format. All animals received clindamycin (prescription solution; clindamycin sulfate; University of Arizona Pharmacy; 30 mg/kg). 1000 spores of C. difficile strain CD630 was used in the challenge studies where indicated, and 108 CFU LAB was used wherever indicated. Group 1 animals received clindamycin on day −3 but no other intervention (black line in
For continuous-dose studies, the clindamycin dose and timing was similar to that above, and only the “C. difficile”, “Empty Vector” and “LAB” groups as above were evaluated. Both “Empty Vector” and Syn-LAB 2.0 (“LAB”) were continuously dosed at 108 CFU per animal per day starting at Day −6 before infection, until death/euthanasia.
Where appropriate, infections commenced 72 hours post-antibiotic administration, and the challenge strain used was C. difficile strain 630 (1000 spores; orally administered in PBS). Animals monitored for disease symptoms (wet-tail, ruffled coat, lethargy) through the course of the studies. Moribund hamsters or those meeting the criteria for euthanasia were administered 270 mg/kg commercial euthanizing solution (Euthanasia III, MedPharma Inc, Pomona, Calif.). Euthanized hamsters were dissected for visualization of gross pathology, and cecal contents harvested and plated on selective medium for recovery and molecular typing of C. difficile (using 16s-23s rDNA intergenic fragment profiling and comparison with the organisms used for infection). In all studies, and all groups, fecal pellets were also collected daily, re-suspended in PBS, homogenized, serially diluted and plated on C. difficile or L. casei selective medium as appropriate.
Immune response studies. For these experiments, age- and weight-matched Golden Syrian hamsters (at least 3 per group) were administered 108 CFU Syn-LAB 2.0 daily, or left untreated, for 21 days. Animals were then euthanized, whole blood harvested via cardiac puncture, and serum immediately retrieved after blood was centrifuged at 1000 g for 10 minutes. This material was aliquoted and stored at −80 C until further use. For immune response assessments, the same methodology as immunoblotting above was used, but serum from Syn-LAB or mock-treated animals was used as the source of primary antibody.
Neonatal piglet studies. All piglet studies were approved by the Institutional Animal Care and Use Committee of the University of Arizona; we assessed Syn-LAB 2.1 safety, tolerability and efficacy in protecting against C. difficile challenge. Newborn male and female piglets were obtained via assisted delivery from a local antibiotic-free, small-volume farm, and transferred to the University of Arizona Central Animal Facility within 2 hours of birth. On Day 2 post-birth, piglets were treated with oral vancomycin (50 mg/kg; prescription solution, University of Arizona Pharmacy) to ablate any pre-existing C. difficile colonization. On day 6 post-birth, piglets were administered 1010 Syn-LAB 2.1 in milk replacer every 8 hours. On day 7, a subset of animals was administered a non-lethal dose of 1000 C. difficile spores of strain 630. Monitoring included checks every 8 hours thereafter, with weight, stimulus response and dehydrations scores recorded. Upon completion of the study, piglets were anaesthetized with Ketamine/Xylazine, and then humanely euthanized with commercial euthanizing solution (Euthanasia III, MedPharma Inc, Pomona, Calif.) followed by cardiac puncture. Histologic analyses included standard hematoxylin-eosin staining of colonic tissues following standard methodologies (Kiernan, 2008, theory and practice, 4th edition), and immunofluorescence staining of tissues with anti-C. difficile SlpA serum as described in detail above for visualization of Syn-LAB 2.0.
Statistical analysis. Multiple statistical tests were employed and utilized the Excel-Stat application to determine significance for experiments involving quantitation. For growth and bacterial burden, Student's t-tests were performed to compute differences between parental and Syn-LAB strains, and errors bars calculated from standard deviation(s). For in vivo studies, Kaplan-Meier survival curves were computed followed by Log-Rank tests for post-hoc analyses.
The results of the experiments are now described.
A 7740 base pair (bp) dual-use vector (pMGM15, SEQ ID NO:1) was chemically synthesized in fragments, and ligated via appropriate restriction enzyme-based biology (See
The precise engineering of this entire 7740 base pair vector was confirmed by complete DNA sequencing.
L. casei (ATCC strain 334) and L. acidophilus (ATCC strain 4356) were tested for purity, confirmed to be the appropriate recipients for Syn-LAB construction via 16S-based speciating PCR, and transformed with the dual use vector PMGM15 (See
SlpA chimera expression was assessed via multiple methodologies for both engineered synthetic biologics. First, fluorescence-activated cell sorting (FACS) was used to determine the degree of heterologous SlpA surface display. In actively growing cultures, almost 100% of bacteria displayed C. difficile SlpA (confirmed by fluorescence shifts in both Syn-LAB 2.0 and Syn-LAB 2.1 compared to the parent strains;
C. difficile-specific SlpA visualization was confirmed to be due to the chimeric protein being incorporated into the lactic acid bacterial cell wall (
Syn-LAB biologics are expected to colonize the mammalian intestine, at least transiently, and likely for longer durations if persistently dosed. To assess any impact on intestinal epithelial cell health and function, a series of studies were performed. First, and to address Syn-LAB's impact on intestinal epithelial barrier function (which is greatly compromised during C. difficile infection), sensitive trans-epithelial electrical resistance flux measurements were performed. In these experiments, confluent human intestinal epithelial cells are grown in a polarized, tightly-organized monolayer such that they exhibit all hallmarks of the healthy gut, including apical and basal faces, and vectorized, or directional, transport of nutrients and solutes. Bacteria are then added to the apical layer, if they perturb the tight para-cellular junctional framework (barrier), electrical resistance is offered by the barrier is lost. This loss of resistance is measured using a sensitive voltmeter, and over time, and provides an indication of the robustness of the gut epithelium. Diarrhea normally occurs when barrier functions is lost, resulting in abnormal fluid transport. Addition of the parent L. casei strain at 100 bacteria/host cell (standard protocol; Wilbur et al., Infection and Immunity, 2015) perturbed barrier over time, and starting as early as 3 hours post-treatment. Interestingly, however, application of Syn-LAB 2.0 protected epithelial cells from any loss of barrier function. Indeed, electrical resistance measurements mimicked, and were statistically indistinguishable, from those observed for un-treated monolayers (
To further assess the impact of prolonged Syn-LAB contact with the host epithelial surface, an in vitro surrogate assay was used to detect host cell death in the presence of the biologic. Human intestinal epithelial cells were grown to confluence, and either mock-treated, or treated with Syn-LAB 2.0, or its parent, for up to 8 hours. Cell death was continuously measured during the treatment by Propidium Iodide (PI) uptake, which stains fragmenting DNA in dead cells, allowing for automated fluorometric measurement. At the end of an 8-hour application, Syn-LAB-treated host cells displayed cell death indistinguishable from mock-treated cells. However, host cells treated with the lactic acid bacterial parent displayed a trend toward increased cell death after 6.5 hours of treatment (
All animal studies were performed in accordance with an approved IACUC protocol (14-526; Vedantam PI). Syn-LAB 2.0 was prepared for in vivo dosing (108-10 bacteria per 200 μL volume per day). Golden Syrian hamsters (6-8 weeks old, 90-110 g weight) were divided into three groups. Group 1 animals received no treatment (control). Group 2 animals received one single dose of the broad-spectrum antibiotic Clindamycin (prescription solution; oral suspension; 30 mg/kg), followed by once-daily dosing of Syn-LAB 2.0 (antibiotic “pre-treatment” group). Group 3 animals received 4 days of Syn-LAB 2.0, followed by one single dose of oral Clindamycin (as above), followed by another 4 days of Syn-LAB 2.0 (antibiotic “mid-cycle treatment” group). Standard hamster chow and water were provided ad libitum. Fecal pellets and weights were collected daily, and pellets were re-suspended in PBS, homogenized, serially diluted and plated on Syn-LAB 2.0 selective medium. In all groups, all animals were devoid of any lactic acid bacterial colonization prior to the start of the study. Groups 2 and 3 animals started shedding Syn-LAB 2.0 on Day 1 post-administration; this continued until the end of the study on Day 8. Group 3 animals that received Clindamycin on Day 4 post-Syn-LAB showed no evidence of the biologic on Day 5, confirming the in vivo susceptibility of Syn-LAB 2.0 to standard antimicrobial therapy (also relevant to biological containment). However, when Syn-LAB feeding was resumed, shedding was also resumed in similar numbers to those observed prior to antimicrobial therapy (representative example shown in
Importantly, all animals were similarly colonized, with Syn-LAB 2.0 fecal titers reaching, or exceeding 105 colony forming units/gram stool on Day 3 post-treatment (
Immunofluorescence studies of colonic tissues harvested post-necropsy revealed dense luminal staining only from Syn-LAB 2.0 treated hamsters (
While the primary goal was to design biologic agents that could competitively occupy C. difficile attachment sites in the gut, we also explored the possibility of an anti-SlpA immune response following long-term Syn-LAB administration. Golden Syrian hamsters were continuously administered Syn-LAB 2.0 as a once-daily 108 CFU dose for 55 days. Hamsters shed the biologic consistently throughout the process confirming that they were appropriately colonized. Age- and weight-matched control hamsters received no treatment. At the end of the study, hamsters were humanely euthanized, whole blood collected, and immunoblot-based analyses performed to assess anti-Syn-LAB immune response. Serum from Syn-LAB 2.0-treated hamsters (
Since single-species probiotics are thought to have limited ability to protect against CDI (Wullt et al, Scand J Infect Dis 35(6-7), 365-367(2003); Vernaya et al., JBI Database System Rev Implement Rep 15(1), 140-164(2017)) we used a combination of Syn-LAB 2.0 and Syn-LAB 2.1 in hamster protection studies. A mixed culture of the biologics (108 CFU total) was administered to antibiotic-sensitized Golden Syrian hamsters either as a fixed dose (FD) formulation (6 doses) or as a continuous dose (CD) formulation (3 days prior to clindamycin until the end of the study). Syn-LAB-treated hamsters were compared to those receiving C. difficile alone, or those administered LAB containing the empty vector. Challenge studies used a high inoculum (˜1000 spores) of C. difficile strain 630 [a virulent, outbreak-associated isolate ((Merrigan et al., PLoS ONE 8, e78404 (2013)).
Fixed dosing of the Syn-LAB combination significantly delayed death of hamsters compared to mock-treated animals, as well as those administered the empty-vector-harboring strains (
Re-administration of clindamycin to Syn-LAB-treated, C. difficile-challenged hamsters 14 days post infection did not result in disease or mortality (not shown). This suggested that Syn-LAB-mediated colonization resistance also ablated C. difficile persistence. Taken together, Syn-LAB administration was highly protective in the hamster model described above.
Preliminary neonatal piglet studies used healthy, newborn animals treated with vancomycin on Day 4 post-farrowing to ensure elimination of any carryover C. difficile bacteria from the farm. Control piglets were given PBS followed by C. difficile challenge, whereas treated piglets were administered three 108 CFU doses of the fast-growing Syn-LAB 2.1 strain over 24 hours. Syn-LAB 2.1 was detected as early as 24 hours after the first dose, and shedding continued until the end of the study (not shown).
In this model, C. difficile infection [1000 spores for piglets (Steele et al, (2010). J Infect Dis 201(3), 428-434), resulted in profuse diarrhea (
This invention provides novel engineered synthetic biologics Syn-LAB 2.0 (L. casei-based), and Syn-LAB 2.1 (L. acidophilus-based) which incorporate comprehensive biological containment features including (1) suicide-competent repA alleles that force chimeric SlpA integration into the genome obviating the requirement for any antibiotic selection; and (2) short-term GI tract viability via irreversible nucleoside auxotrophy. These biologics target a CDC “Urgent Category” pathogen (C. difficile), focus on a non-traditional therapeutics that avoids the use of antibiotics, and are rooted in a “bedside-to-bench-to-bedside” paradigm. Importantly, three mechanisms which have been proposed as being crucial to C. difficile infection prevention are all hallmarks of Syn-LAB 2.0 and Syn-LAB 2.1, namely (1) provision of robust colonization resistance, (2) potential for adaptive immune response to SlpA, an essential CD antigen, and (3) accelerated endogenous microbiota recovery via use of a safe non-antibiotic intervention (thereby reducing risk of CDI relapse).
Appropriate Syn-LAB 2.0 and Syn-LAB 2.1 dosing may confer the advantage of long-term protection against CD colonization by inducing an anti-SlpA immune response. LAB, especially L. casei, induce both mucosal and system immune response(s) (Campos et al, Microbes Infect 10, 481-488 (2008)). CD SlpA avidly binds to the surface epithelium and exposed lamina propria in GI tissues (Calabi et al, Infect Immun 70, 5770-5778 (2002)). The specific subunit used herein has also been shown to be antigenic, reacting with sera from CDI patients (Spigaglia et al, J Med Microbiol 60, 1168-1173 (2011)). Importantly, an anti-SlpA response is expected to be sustained across diverse CD molecular types since the construct of this invention was specifically engineered to encode a highly-conserved SlpA domain. Further, SlpA itself is essential; therefore, escape mutants are unlikely. Previous studies on immunization with CD surface-layer proteins (SLPs) included mixtures of multiple SLP antigens (>15), yielding inconsistent results in CDI prevention. The approach followed here, based only on SlpA, the most adherent CD SLP (Ni Eidhin et al, FEMS Immunol Med Microbiol 52, 207-218 (2008); Pechine et al, FEMS Immunol Med Microbiol 63, 73-81 (2011)), is more likely to prove immunologically robust.
Syn-LAB 2.0 and Syn-LAB 2.1 are synthetic biologic agents wherein the lactic acid bacteria Lactobacillus casei and Lactobacillus acidophilus, respectively, have been engineered to stably and constitutively express the major Clostridium difficile adhesin SlpA on their cell-surface. Both agents harbor suicide plasmids expressing a codon optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly-adherent, host-cell-binding domain of Clostridium difficile Surface-Layer Protein A (SlpA; the major adhesin). Both agents also contain built-in and engineered biocontrol, in that (1) the plasmid is lost at body temperature unless stably integrated into the chromosome, obviating the need for any antibiotic selection; and (2) the whole strain is lost within a few days at body temperature due to an engineered nucleoside auxotrophy. Both agents also contain a fluorescent, non-toxic allele (ytvA) that allows for selection of the agent under blue-light visualization, and is thus an easy surrogate for in vitro or in vivo detection. Syn-LAB 2.0 and Syn-LAB 2.1 both possess positive biophysical and in vivo properties compared with their lactic acid bacterial antecedents: (1) they both adhere robustly to human intestinal epithelial cells in vitro; and (2) they both strongly and constitutively display the SlpA chimera on their cell surface(s). In addition, Syn-LAB 2.0 also (3) protects human intestinal epithelial barrier function in vitro; (4) is safe, tolerable and palatable to Golden Syrian hamsters at high daily doses; and (5) is detectable in hamster feces within 24 hours of dosing, indicating robust colonization. Most importantly, Syn-LAB 2.0 is rapidly lost from the hamster GI tract unless continuously dosed, or upon antibiotic administration, suggesting that the presently engineered bio-containment is fully functional.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
The present application is a Section 371 U.S. national stage entry of International Patent Application No. PCT/US2018/052656, International Filing Date Sep. 25, 2018 which is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/563,365, filed Sep. 26, 2017 which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant No. AI 121590 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/052656 | 9/25/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/067447 | 4/4/2019 | WO | A |
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| 2016118900 | Jul 2016 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20200239527 A1 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62563365 | Sep 2017 | US |
