Recombinant Lactococcus lactis expressing Escherichia coli colonization factor antigen I (CFA/I) fimbriae and their methods of use
Inventors
Pascual, David W. • Maddaloni, Massimo
Assignees
Montana State University Bozeman
Publication Number
US-9931390-B2
Publication Date
2018-04-03
Expiration Date
2033-09-24
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Abstract
The present disclosure relates generally to therapeutic compositions comprising recombinant bacteria. Further, the disclosure elaborates upon methods of utilizing the taught therapeutic compositions to treat autoimmune and inflammatory disease. The present teachings also relate to the disclosed recombinant bacteria and methods of producing the recombinant bacteria utilized in the compositions and methods. Further taught herein are dietary supplements and food additive compositions comprising the taught recombinant bacteria.
Core Innovation
The invention provides therapeutic compositions comprising recombinant Gram-positive bacteria, specifically Lactococcus lactis, that are engineered to express enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae. The invention also encompasses methods of using these compositions to treat autoimmune and inflammatory diseases. Additionally, it covers methods for the production of the disclosed recombinant bacteria and their incorporation into dietary supplements and food additive compositions.
Prior to this disclosure, there had not been a successful method for expressing ETEC CFA/I fimbriae in Gram-positive bacteria. The problem addressed is the need for safer and more effective bacterial-based therapeutics and vaccines that are not based on attenuated Gram-negative strains such as Salmonella, which present drawbacks such as the presence of endotoxin (LPS), risk of reversion to virulence, complications from pre-existing immunity, and horizontal gene transfer hazards.
By successfully engineering Gram-positive vectors, particularly Lactococcus lactis, to express ETEC CFA/I fimbriae, the present invention demonstrates and teaches compositions and methods that modulate immune responses to treat or prevent autoimmune and inflammatory diseases. The compositions may be included in pharmaceutically acceptable carriers, combined with foodstuffs and supplements, and delivered through various routes to beneficially influence immune function.
Claims Coverage
The patent contains multiple independent claims covering synthetic genetic constructs, recombinant host cells, compositions, and methods. The main inventive features are outlined below.
Composite promoter polynucleotide construct
A polynucleotide sequence comprising a composite promoter sequence consisting of: - a nisin promoter, - a p170 promoter, and - a cp25 promoter, where at least one promoter is modified to improve mRNA stability, and one or more genes are under the control of the composite promoter.
Signal sequence integration and Shine-Dalgarno use
Incorporation of in-frame replacement of native signal sequences in the one or more genes by signal sequences selected from usp45, Exp4, lac, and prtP, and use of at least one Shine-Dalgarno sequence in the polynucleotide sequence (with embodiments specifying two or more Shine-Dalgarno sequences).
Defined composite promoter sequence
The composite promoter sequence is further defined by SEQ ID NOs: 6-8 as contained in SEQ ID NO:1, or by sharing at least 90–95% identity with these sequences. Each promoter (nisin, p170, cp25) shares at least 95% sequence identity with the corresponding SEQ ID numbers.
Recombinant prokaryotic host cell expressing the construct
A prokaryotic host cell, especially a Gram-positive bacterium, preferably lactic acid bacterium or specifically Lactococcus lactis, comprising the above polynucleotide sequence and expressing the one or more genes.
Compositions including recombinant host cells
A composition comprising the recombinant host cell described above, wherein the composition may further comprise a foodstuff or a food supplement.
Methods for gene expression in prokaryotic hosts
A method for expressing one or more genes by introducing and expressing the described polynucleotide sequence with a composite promoter in a prokaryotic host cell. The one or more genes may be prokaryotic or eukaryotic in origin.
Method of administration of recombinant compositions
A method of administering a composition by ingestion, where the composition comprises the recombinant host cell as detailed above.
Polynucleotide with at least two defined promoters
A polynucleotide sequence comprising a composite promoter with at least two of the following: a nisin promoter, a p170 promoter, and a cp25 promoter, each sharing at least 95% sequence identity with the corresponding SEQ ID NOs, and one or more genes under the control of the promoter, with at least one promoter modified for mRNA stability.
The inventive features include engineered composite promoters with improved mRNA stability, replacement of signal sequences for optimal expression in Gram-positive hosts, specifically lactic acid bacteria, compositions including engineered host cells, and novel methods for gene expression and administration relevant to therapeutic and food supplement uses.
Stated Advantages
Use of Gram-positive lactic acid bacteria avoids the presence of lipopolysaccharide (LPS) toxins found in Gram-negative vectors like Salmonella.
Lactic acid bacteria, such as Lactococcus lactis, are Generally Recognized as Safe (GRAS) and do not pose a risk of reversion to a virulent state.
There is an insignificant risk of horizontal gene transfer to invasive bacteria when using Gram-positive vectors compared to Salmonella-based systems.
Lactic acid bacteria have a history of beneficial association with human intestinal flora and can act synergistically with probiotics.
The recombinant Lactococcus lactis expressing ETEC CFA/I fimbriae demonstrates increased potency and efficacy in preventing autoimmune diseases as compared to Salmonella vectors.
The vector is minimally immunogenic, allowing for multiple dosing or instillation without eliciting a major negative immune response.
Compositions are suitable for oral, nasal, or other mucosal administration and can be incorporated into foodstuffs, dietary supplements, and probiotics.
Documented Applications
Prevention and treatment of autoimmune and inflammatory diseases by administering recombinant Gram-positive bacteria expressing ETEC CFA/I fimbriae.
Use in dietary supplements and food additive compositions to support a healthy immune system or supplement an individual's normal dietary regime.
Prophylactic and therapeutic administration for conditions like rheumatoid arthritis (using collagen-induced arthritis model) and multiple sclerosis (using experimental autoimmune encephalomyelitis model).
Formulation with pharmaceutically acceptable carriers for delivery as a foodstuff or food supplement, including beverages, dairy products, yogurts, fermented foods, powdered compositions, and encapsulated or liquid forms.
Stimulation of anti-inflammatory cytokines (IL-10, TGF-β) and suppression of proinflammatory cytokines (IFN-γ, TNF-α, IL-17) in a subject treated with the disclosed recombinant bacteria.
Eliciting immune regulation by increasing Treg cell induction in humans upon administration of the recombinant Lactococcus lactis expressing ETEC CFA/I fimbriae.
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