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-9452205-B2
Publication Date
2016-09-27
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 Lactococcus lactis bacterial cells that express enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae. The recombinant bacteria are engineered to include nucleotide sequences coding for the CFA/I fimbriae structural genes cfaA, cfaB, cfaC, and cfaE, which are expressed within the Lactococcus lactis cells, with the sequences optionally codon-optimized and arranged in a rearranged order for enhanced expression.
This innovation addresses the need for safer and more effective bacterial-based therapeutics for treating autoimmune and inflammatory diseases. Prior art relied on attenuated Gram-negative Salmonella vectors to deliver CFA/I fimbriae, which posed risks such as endotoxin-mediated inflammation, reversion to virulence, potential for horizontal gene transfer, and issues with pre-existing immunity. There was no reported successful expression of E. coli CFA/I fimbriae in Gram-positive bacteria prior to this disclosure.
By employing a Generally Recognized as Safe (GRAS) Gram-positive bacterium, such as Lactococcus lactis, to express and deliver the CFA/I fimbriae, the invention overcomes these drawbacks. The compositions are designed for use as therapeutics, dietary supplements, or food additives for the prophylactic or therapeutic treatment of autoimmune and inflammatory diseases, providing an alternative delivery platform that combines safety and immunomodulatory potential.
Claims Coverage
The patent contains several independent claims that cover different aspects of the invention. The principal inventive features are summarized below.
Therapeutic composition comprising recombinant Lactococcus lactis expressing CFA/I fimbriae
A composition for the treatment of an autoimmune or inflammatory disease, comprising: - A recombinant Lactococcus lactis bacterial cell containing nucleotide sequences coding for the enterotoxigenic Escherichia coli colonization factor antigen I fimbriae genes cfaA, cfaB, cfaC, and cfaE. - Expression of the abovementioned genes (with cfaA as SEQ ID NO: 3, cfaB as SEQ ID NO: 2, cfaC as SEQ ID NO: 4, cfaE as SEQ ID NO: 5). - An acceptable carrier.
Composition with polynucleotide sequence sharing at least 95% identity with SEQ ID NO: 1
A composition for the treatment of an autoimmune or inflammatory disease comprising: - A recombinant Lactococcus lactis bacterial cell containing a nucleotide sequence coding for CFA/I fimbriae genes cfaA, cfaB, cfaC, and cfaE, - Wherein the nucleotide sequence comprises a polynucleotide sequence sharing at least 95% sequence identity with SEQ ID NO: 1.
Recombinant Lactococcus lactis bacterial cell expressing CFA/I fimbriae genes
A recombinant Lactococcus lactis bacterial cell comprising: - Nucleotide sequences coding for E. coli colonization factor antigen I fimbriae genes cfaA, cfaB, cfaC, and cfaE, - Expression of the CFA/I fimbriae genes (with nucleotide sequences corresponding to SEQ ID NO: 2, 3, 4, and 5 for respective genes).
Recombinant Lactococcus lactis bacterial cell with nucleotide sequence sharing at least 95% identity with SEQ ID NO: 1
A recombinant Lactococcus lactis bacterial cell comprising: - A nucleotide sequence coding for CFA/I fimbriae genes cfaA, cfaB, cfaC, and cfaE, - The nucleotide sequence comprises a polynucleotide sequence sharing at least 95% sequence identity with SEQ ID NO: 1.
Method for producing a composition for treatment of autoimmune or inflammatory disease
A method comprising: 1. Introducing a nucleotide sequence coding for E. coli colonization factor antigen I fimbriae genes cfaA, cfaB, cfaC, and cfaE (comprising a polynucleotide sequence sharing at least 95% identity with SEQ ID NO: 1) into a recipient Lactococcus lactis bacterial cell. 2. Optionally, further comprising culturing the bacterial cell under conditions which allow for expression of the fimbriae.
The inventive features encompass the composition and methods involving recombinant Lactococcus lactis expressing E. coli CFA/I fimbriae for treating autoimmune and inflammatory diseases, including specific genetic constructs and methods for their production.
Stated Advantages
The recombinant lactic acid bacteria do not present endotoxin-associated inflammation, as they lack LPS found in Gram-negative vectors such as Salmonella.
Lactococcus lactis has a GRAS (Generally Recognized as Safe) status, eliminating concerns of reversion to virulence present in Salmonella-based systems.
The disclosed vectors show minimal risk of horizontal gene transfer to invasive bacteria, unlike Salmonella vectors.
Lactic acid bacteria vectors do not elicit a significant anti-fimbrial immune response, permitting repeated dosing.
Compositions are more potent in suppressing proinflammatory cytokines and increasing anti-inflammatory cytokines than Salmonella-based systems.
The compositions may be combined with pharmaceutically acceptable carriers or foodstuffs, providing flexible, consumer-friendly delivery vehicles.
The recombinant bacteria support a healthy immune system and act as probiotics or dietary supplements.
Documented Applications
Treatment or prevention of autoimmune diseases, specifically rheumatoid arthritis as modeled by collagen-induced arthritis in mice.
Treatment or prevention of inflammatory diseases, including experimental autoimmune encephalomyelitis, a model for multiple sclerosis.
Use as a probiotic composition to support a healthy immune system.
Use in dietary supplements or food additives containing recombinant lactic acid bacteria expressing CFA/I fimbriae.
Method for suppressing proinflammatory cytokines (such as IFN-γ, TNF-α, IL-17) and increasing anti-inflammatory cytokines (IL-10, TGF-β) in a subject.
Administration in forms such as beverages, dairy products, yogurts, or encapsulated and powdered supplements.
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