Live, oral vaccine for protection against Shigella dysenteriae serotype 1
Inventors
Kopecko, Dennis J. • Xu, De-Qi
Assignees
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Publication Number
US-9402889-B2
Publication Date
2016-08-02
Expiration Date
2025-05-24
Abstract
The invention relates to Salmonella typhi Ty21a comprising core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide (O-Ps) and DNA encoding O antigen biosynthesis, said DNA selected from the group consisting of: a) the DNA sequence set out in any one of SEQ ID NOs: 1 and 2 and species homologs thereof; b) DNA encoding Shigella dysenteriae serotype 1 polypeptides encoded by any one of SEQ ID NOs: 1 and 2, and species homologs thereof; and c) DNA encoding a O antigen biosynthesis gene product that hybridizes under moderately stringent conditions to the DNA of (a) or (b); and related sequences, compositions of matter, vaccines, methods of using, and methods of making.
Core Innovation
The invention relates to an attenuated strain of Salmonella typhi Ty21a that expresses core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide (O-Ps) and includes DNA encoding O antigen biosynthesis. The DNA comprises sequences set out in SEQ ID NOs: 1 and 2 and related species homologs, encoding Shigella dysenteriae serotype 1 polypeptides necessary for O antigen biosynthesis. The invention also covers related sequences, compositions, vaccines, methods of use, and methods of making such strains.
Shigella dysenteriae serotype 1 causes a severe form of bacillary dysentery with high mortality rates worldwide, especially in children and developing countries. There are currently no FDA-licensed vaccines against Shigella, and the disease's management complexity is increased by multi-drug resistant isolates. Protective immunity correlates with antibodies against the O-specific polysaccharide (O-Ps) present on the lipopolysaccharide (LPS) of the bacteria. Prior attempts at vaccine development faced challenges such as plasmid instability and insufficient protection. The invention addresses this by cloning and sequencing essential genes (rfb and rfp) responsible for O-Ps biosynthesis from Shigella dysenteriae serotype 1 strain 1617 and constructing a genetically stable plasmid that expresses core-linked O-Ps in Salmonella typhi Ty21a, resulting in an oral vaccine candidate.
The invention includes the molecular characterization of nine open reading frames (ORFs) from the rfb locus and the rfp gene on a small plasmid, all necessary for O-Ps biosynthesis. A low copy vector (pGB2) carrying both loci was constructed to produce surface-expressed, core-linked Shigella dysenteriae serotype 1 O-Ps in transformed Salmonella typhi and Escherichia coli. The expressed O-Ps exhibits a typical ladder pattern similar to the wild-type Shigella strain. This stable expression in a live, attenuated bacterial vector is anticipated to provide an effective oral vaccine against this severe dysentery-causing pathogen.
Claims Coverage
The patent contains two independent claims directed to attenuated bacterial strains expressing core-linked Shigella dysenteriae serotype 1 O-Ps and DNA encoding O antigen biosynthesis. The following inventive features are identified from these claims.
Attenuated strain comprising core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide and DNA encoding O antigen biosynthesis
An attenuated strain of Salmonella comprising core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide (O-Ps) and DNA encoding O antigen biosynthesis. The DNA is selected from (a) sequences set out in SEQ ID NOs: 1 and 2 and homologous species nucleic acids sharing at least about 90% identity; (b) DNA encoding Shigella dysenteriae serotype 1 polypeptides encoded by SEQ ID NOs: 1 and 2 and homologous species nucleic acids sharing at least about 90% identity; and (c) DNA encoding variants of these polypeptides comprising O antigen biosynthesis polypeptides with amino acid deletions, replacements, or additions that do not diminish O antigen or protective immunological activity.
DNA localization and promoter control in the attenuated strain
The DNA encoding O antigen biosynthesis is present on a plasmid and is under the control of its cognate promoters, enabling stable and regulated expression in the attenuated strain.
Attenuated strain comprising core-linked O-Ps and DNA encoding O antigen biosynthesis in Shigella
An attenuated strain of Shigella comprising core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide (O-Ps) and DNA encoding O antigen biosynthesis with the same DNA sequence and variant definitions as the Salmonella strain. This claim specifically encompasses Shigella species such as Shigella flexneri and Shigella sonnei.
The independent claims cover attenuated Salmonella and Shigella strains expressing core-linked Shigella dysenteriae serotype 1 O-specific polysaccharide through DNA encoding O antigen biosynthesis comprising sequences of SEQ ID NOs: 1 and 2 or variants thereof, with the DNA localized on plasmids under cognate promoters, enabling stable and immunogenically effective expression.
Stated Advantages
The invention provides a genetically stable expression system of Shigella dysenteriae serotype 1 O antigen in live attenuated Salmonella typhi Ty21a, promoting effective oral vaccine development.
It enables the production of core-linked O-specific polysaccharide resembling native Shigella LPS, which is anticipated to stimulate protective immunity.
The vaccine approach addresses limitations of prior unstable plasmid constructs and low protection efficiency seen in earlier studies.
Documented Applications
Use as a live, oral vaccine for the prophylactic or therapeutic treatment of bacterial infection caused by Shigella dysenteriae serotype 1.
Development of vaccine strains expressing heterologous O-specific polysaccharide genes for immunization against Gram-negative enteric pathogens.
Potential enhancement of vaccine strains to include additional antigens such as Shiga toxin B subunit to provide broader protection.
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