Multifunctional oral vaccine based on chromosome recombineering
Inventors
Dharmasena, Madushini Nirosha • Kopecko, Dennis J.
Assignees
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Publication Number
US-9750793-B2
Publication Date
2017-09-05
Expiration Date
2033-09-16
Abstract
A recombineered Salmonella typhi Ty21a, compositions and vaccines comprising such a Ty21a, and a method for recombineering comprising inserting a large antigenic region into a bacterial chromosome for the purpose of making multivalent vaccines to protect against one or more disease agents are described herein.
Core Innovation
The invention provides a recombineered Salmonella Typhi Ty21a strain with heterologous Shigella O-antigen biosynthetic gene regions stably integrated into the Ty21a chromosome. Specifically, minimal essential gene regions encoding Shigella sonnei, Shigella dysenteriae serotype 1, Shigella flexneri 2a, and Shigella flexneri 3a O-antigens are cloned and integrated into the nonfunctional Vi capsule locus of Ty21a, enabling stable, antibiotic marker-free expression of heterologous O-antigens concurrently with or without homologous Salmonella Typhi O-antigen expression. This results in a multifunctional oral live vaccine candidate that elicits immune protection against both enteric fever caused by Salmonella Typhi and shigellosis caused by the respective Shigella species.
The problem addressed is the lack of a licensed, genetically stable multivalent vaccine protecting against the major causes of shigellosis and enteric fevers. Previous plasmid-based expression of large Shigella O-antigen biosynthetic gene clusters in Ty21a showed instability, especially upon removal of antibiotic resistance markers required for human use, posing safety and regulatory limitations. Thus, effective and stable chromosomal integration methods were needed to insert large antigenic gene regions into a bacterial chromosome without loss or instability and without retaining antibiotic resistance markers, to produce safe and efficacious multivalent live oral vaccines.
Claims Coverage
The patent includes multiple independent claims covering recombinant Salmonella Typhi Ty21a strains expressing heterologous Shigella O-antigen gene regions, a plasmid construct for cloning these regions, and a method for recombineering large antigenic gene regions into bacterial chromosomes. Five main inventive features are detailed.
Stable expression of Shigella sonnei O-antigen genes in Salmonella Typhi Ty21a chromosome
A Salmonella Typhi Ty21a strain comprising a Shigella sonnei form 1 O-antigen biosynthetic gene region inserted into the chromosome such that heterologous S. sonnei O-antigen is stably expressed with or without homologous S. Typhi O-antigen, eliciting immune protection against virulent S. sonnei and S. Typhi challenge. The gene region includes at least about 90% sequence identity to SEQ ID NO:2.
Inclusion of additional O-antigen regions from diverse bacteria in Ty21a
The Salmonella Typhi Ty21a strain further includes O-antigen biosynthetic gene regions from bacterial strains in groups including Shigella species, Escherichia coli serotypes, Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacter species, Yersinia species, Plesiomonas species, and Pseudomonas species.
Stable expression of Shigella dysenteriae serotype 1 O-antigen genes in Ty21a chromosome
A Salmonella Typhi Ty21a strain with Shigella dysenteriae 1 O-antigen biosynthetic gene region inserted into its chromosome, stably expressing the heterologous O-antigen with or without S. Typhi homologous O-antigen, eliciting immune protection against virulent S. dysenteriae and S. Typhi challenge. The gene region includes at least about 90% sequence identity to SEQ ID NO:33.
A plasmid construct enabling insertion of large antigenic gene regions into chromosome
A plasmid construct containing a DNA sequence as set forth in SEQ ID NO:1 or sharing at least 90% sequence identity, comprising a genetically selectable marker flanked by FRT sites, a multiple cloning site, and flanking two chromosome homology sites, designed for recombineering of large antigenic gene regions.
A recombineering method for large antigenic gene region insertion into bacterial chromosome
A method comprising i) cloning a large antigenic region into a vector with a selectable marker flanked by FRT sites and flanking chromosome homology arms, ii) integrating the region into the bacterial chromosome using λ Red recombination, iii) selecting for the marker, and iv) removing the selectable marker, enabling stable chromosomal integration of antigenic regions typically about 5 to 20 kb long, notably including Shigella sonnei, dysenteriae, and flexneri O-antigen genes in Salmonella Typhi Ty21a.
The claimed invention encompasses stable chromosomal integration of large heterologous O-antigen gene regions from Shigella species into Salmonella Typhi Ty21a to achieve stably expressed multifunctional oral vaccine strains. It also covers plasmid constructs and recombineering methods enabling such stable insertions and subsequent removal of antibiotic selection markers.
Stated Advantages
The strain Ty21a-Ss with chromosomally integrated Shigella sonnei O-antigen genes exhibits 100% genetic stability without the need for continuous antibiotic selection.
Stable and simultaneous expression of heterologous Shigella O-antigen and homologous Salmonella Typhi O-antigen elicits robust immune protection against both pathogens in animal models.
The recombineering technique enables insertion of large gene regions (>11 kb) into bacterial chromosomes efficiently, improving vaccine genetic stability and safety by removing antibiotic markers.
The method is applicable to multiple bacterial genera and can be used for constructing multivalent vaccines targeting different Shigella serotypes and potentially other bacterial pathogens.
Documented Applications
Development of a live, orally administered multivalent vaccine to protect against enteric fevers caused by Salmonella Typhi and shigellosis caused by various Shigella species including S. sonnei, S. dysenteriae 1, S. flexneri 2a, and S. flexneri 3a.
Use of chromosomally integrated Shigella O-antigen biosynthetic genes in Salmonella Typhi Ty21a as a platform to biologically manufacture protein-LPS conjugate products for vaccine development.
Insertion of large antigenic regions into chromosomes of enteric bacteria for the production of genetically stable vectored vaccines utilizing live attenuated Salmonella, Shigella, Listeria, Yersinia, Escherichia coli, Enterobacteriaceae, or protozoan strains.
Administration of the recombinant Ty21a strains as vaccines via oral, intranasal, intraperitoneal, or other routes to prevent or treat bacterial infections such as typhoid fever and shigellosis.
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