Multifunctional oral vaccine based on chromosome recombineering

A chromosome and gene technology, applied in multivalent vaccines, genetic engineering, vaccines, etc., can solve problems such as anti-antibiotic resistance markers, plasmid instability, etc.

Inactive Publication Date: 2015-07-29
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The resulting plasmid encoding Shigella LPS is expected to be stable for more than 50 growth generations in non-selective media, but it still contains objectionable antibiotic resistance markers
Deletion of this antibiotic resistance marker results in pronounced plasmid instability

Method used

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  • Multifunctional oral vaccine based on chromosome recombineering
  • Multifunctional oral vaccine based on chromosome recombineering
  • Multifunctional oral vaccine based on chromosome recombineering

Examples

Experimental program
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Embodiment approach

[0077] In one embodiment, the invention includes a prokaryotic microorganism. In another embodiment, the prokaryotic microorganism is a bacterial species. Preferably, said prokaryotic microorganism is an attenuated strain of Salmonella. However, other prokaryotic microorganisms such as attenuated Escherichia coli, Shigella, Yersinia, Lactobacillus, Mycobacteria, Listeria or Vibrio Strains are also encompassed by the invention. Exemplary suitable microbial strains include, but are not limited to, Salmonella typhimurium, Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis, Escherichia coli, Shigella flexneri, Shigella sonnei , Vibrio cholerae (Yamamoto, et al. (2009) Gene 438:57-64), Pseudomonas aeruginosa (Lesic, et al. (2008) BMC Mol Biol 9:20), Yersinia pestis Yersinia pestis (Sun, et al. (2008) Applied and Env Microbiol 74:4241-4245) and Mycobacterium bovis (BCG). Notably, λred does not function in mycobacteria, but another phage (e.g. Che9c gp61) has been us...

Embodiment 1

[0170] Example 1: Cloning of minimal essential Shigella sonnei O-antigen biosynthesis genes

[0171] A large 30 kb region and flanking sequences encoding the biosynthesis of the S. sonnei type I O-antigen were pre-identified (initially by deletion analysis) to determine the minimal essential sequence for O-antigen expression. Together with DNA sequence analysis of this region, these deletion data indicated the presence of a putative promoter and 10 orf( figure 1 A contiguous 12.3 kb region in orf 4-13) is required for O-antigen biosynthesis in Shigella sonnei. These deletion studies also suggest that wzz (ofr3), which is typically involved in the regulation of O-antigen chain length, is not required for type 1 expression in Ty21a, but the latter part of wzz apparently contains the putative type 1 O-antigen biosynthesis operon promoter ( Xu, et al. (2002) Infect and Immun 70:4414-4423.

[0172] Construction of pMD-TV plasmid

[0173] Cloning is performed using standard mol...

Embodiment 2

[0182] Example 2: Integration of linear Shigella DNA into the Ty21a chromosome

[0183] Datsenko and Wanner (Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA, 97:6640-6645) previously described a method based on the highly efficient lambda phage red recombination system, which enables Antibiotic resistance gene recombination replaces E. coli chromosomal sequences to generate specifically targeted gene mutations, generated by PCR using primers containing a 36-50 bp extension homologous to the targeted mutated gene. The λred system includes β, γ and exo genes, the products of which are called Beta, Gam and Exo, respectively (Murphy (1998) J. Bacteriol., 180:2063-2071). Gam inhibits the host RecB, C, D exonuclease and SbcC, D nuclease activities, resulting in exogenously added linear DNA is not degraded. The Exo protein is a dsDNA-dependent exonuclease that binds the ends of each strand while degrading the other strand in the 5' to 3' direction. Beta binds the resulting ssDN...

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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.

Description

[0001] Related application materials [0002] Each application and patent cited herein, and each document or reference cited in each application and patent (including during the prosecution of each issued patent; an "Application Cited Document") and corresponding to and / or asserting that any Each of the PCT and foreign applications or patents to which these applications and patents claim priority, and each document cited or referred to in each application's cited documents, is hereby incorporated by reference and may be used in the practice of the present invention. More generally, a document or reference is listed in a reference that precedes a claim or is cited in the text; each of these documents or references (herein-listed references) and each of the documents or references listed in each of the herein-listed references Each document or reference (including any manufacturer's instructions, instructions, etc.) is incorporated herein by reference. [0003] Statement of Gover...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31A61K35/74A61P31/04A61K39/00
CPCC07K14/25A61K39/0275A61K2039/70A61K39/0283A61K2039/523A61K2039/542A61P31/04Y02A50/30A61K2039/522C07K14/255C07K2319/40C12N1/20
Inventor M·N·达马塞纳D·J·科佩奇科
Owner UNITED STATES OF AMERICA
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