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Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

Karlsson SL, Ax E, Nygren E, Källgård S, Blomquist M, Ekman A, Benktander J, Holmgren J, Lebens M - PLoS ONE (2014)

Bottom Line: The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria.Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine.This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology at Institute of Biomedicine, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

ABSTRACT
We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

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Related in: MedlinePlus

Colony blot results.O1 Inaba V. cholerae strain JS1569 was transformed with expression plasmids carrying different mutant wbeT genes. Different mutations in the wbeT gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the wbeT genes sequenced. The mutations present in the different clones are indicated.
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pone-0108521-g001: Colony blot results.O1 Inaba V. cholerae strain JS1569 was transformed with expression plasmids carrying different mutant wbeT genes. Different mutations in the wbeT gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the wbeT genes sequenced. The mutations present in the different clones are indicated.

Mentions: We determined that a point mutation in wbeT creating the mutation S158P in the gene product completely destroyed its LPS methylation activity thus resulting in the Inaba serotype. We hypothesized that this site is important for the activity of the wbeT–encoded protein and therefore could be used as a target site for directed mutagenesis in order to generate mutants with reduced activity of the wbeT gene product. A mutation library was constructed in the classical Inaba strain JS1569 as described in the Material and Methods. Fifty different colonies from the mutation library were tested using antibody–based colony blotting. It was clear that the colonies expressed different amounts of Ogawa LPS reflected in different staining intensity from nil to strong. Selected clones from the plate were further analyzed by sequencing the wbeT gene in their plasmids. A typical blot is shown in Figure 1 together with the sequence of selected clones. Twenty–eight additional plasmids were sequenced and besides plasmids carrying the wild–type serine the following substitutions were identified at amino acid 158; A,V,L,I,F,W,M,P,G,T,C,R, and stop codons. Interestingly, no highly polar amino acids were found at position 158 other than a single clone encoding for arginine.


Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

Karlsson SL, Ax E, Nygren E, Källgård S, Blomquist M, Ekman A, Benktander J, Holmgren J, Lebens M - PLoS ONE (2014)

Colony blot results.O1 Inaba V. cholerae strain JS1569 was transformed with expression plasmids carrying different mutant wbeT genes. Different mutations in the wbeT gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the wbeT genes sequenced. The mutations present in the different clones are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232259&req=5

pone-0108521-g001: Colony blot results.O1 Inaba V. cholerae strain JS1569 was transformed with expression plasmids carrying different mutant wbeT genes. Different mutations in the wbeT gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the wbeT genes sequenced. The mutations present in the different clones are indicated.
Mentions: We determined that a point mutation in wbeT creating the mutation S158P in the gene product completely destroyed its LPS methylation activity thus resulting in the Inaba serotype. We hypothesized that this site is important for the activity of the wbeT–encoded protein and therefore could be used as a target site for directed mutagenesis in order to generate mutants with reduced activity of the wbeT gene product. A mutation library was constructed in the classical Inaba strain JS1569 as described in the Material and Methods. Fifty different colonies from the mutation library were tested using antibody–based colony blotting. It was clear that the colonies expressed different amounts of Ogawa LPS reflected in different staining intensity from nil to strong. Selected clones from the plate were further analyzed by sequencing the wbeT gene in their plasmids. A typical blot is shown in Figure 1 together with the sequence of selected clones. Twenty–eight additional plasmids were sequenced and besides plasmids carrying the wild–type serine the following substitutions were identified at amino acid 158; A,V,L,I,F,W,M,P,G,T,C,R, and stop codons. Interestingly, no highly polar amino acids were found at position 158 other than a single clone encoding for arginine.

Bottom Line: The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria.Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine.This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology at Institute of Biomedicine, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

ABSTRACT
We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

Show MeSH
Related in: MedlinePlus