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Bacterial antigen expression is an important component in inducing an immune response to orally administered Salmonella-delivered DNA vaccines.

Gahan ME, Webster DE, Wesselingh SL, Strugnell RA, Yang J - PLoS ONE (2009)

Bottom Line: Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology.The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter.These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful oral delivery of C-fragment DNA vaccines containing the CMV promoter by S. typhimurium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. michelle.gahan@canberra.edu.au

ABSTRACT

Background: The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contributions of prokaryotic and eukaryotic expression in production of the vaccine antigen. Gene expression in DNA vaccines is commonly under the control of the eukaryotic cytomegalovirus (CMV) promoter. The aim of this study was to identify and disable putative bacterial promoters within the CMV promoter and evaluate the immunogenicity of the resulting DNA vaccine delivered orally by S. typhimurium.

Methodology/principal findings: The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter. These promoters have homology to the bacterial consensus sequence and functional activity. To disable prokaryotic expression from the CMV promoter a series of genetic manipulations were performed to remove the two major bacterial promoters and add a bacteria transcription terminator downstream of the CMV promoter. S. typhimurium was used to immunise BALB/c mice orally with a DNA vaccine encoding the C-fragment of tetanus toxin (TT) under control of the original or the modified CMV promoter. Although both promoters functioned equally well in eukaryotic cells, as indicated by equivalent immune responses following intramuscular delivery, only the original CMV promoter was able to induce an anti-TT specific response following oral delivery by S. typhimurium.

Conclusions: These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful oral delivery of C-fragment DNA vaccines containing the CMV promoter by S. typhimurium.

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Location of S. typhimurium promoter sequences within the CMV promoter.(A) DNA sequence of pcDNA3/Cfrag from the start of the CMV promoter highlighting the location of the two primary bacterial promoters (blue), the regulatory regions of the CMV promoter (red) and the start codon of the C-fragment gene (underlined). (B) Alignment of the −35 and −10 promoter regions of the two primary S. typhimurium promoters with the bacterial promoter consensus sequence. The TGT enhancer region located within promoter 2 is shown in bold.
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pone-0006062-g003: Location of S. typhimurium promoter sequences within the CMV promoter.(A) DNA sequence of pcDNA3/Cfrag from the start of the CMV promoter highlighting the location of the two primary bacterial promoters (blue), the regulatory regions of the CMV promoter (red) and the start codon of the C-fragment gene (underlined). (B) Alignment of the −35 and −10 promoter regions of the two primary S. typhimurium promoters with the bacterial promoter consensus sequence. The TGT enhancer region located within promoter 2 is shown in bold.

Mentions: The transcription initiation sites of the bacterial promoter sequences were identified by primer extension analysis using RNA extracted from BRD509 (pcDNA3/Cfrag) and BRD509. Two strong primary bacterial transcription initiation sites situated within the CMV promoter and multiple minor promoters were identified (figure 2). The first of these (promoter one, P1) is located 428 base pairs (bp) upstream of the AP1 region of the CMV promoter and the second promoter (promoter two, P2) is 292 bp upstream of AP1. Both bacterial promoters are located upstream of the CMV promoter regulatory elements and the C-fragment start codon (figure 3a). Comparison of the −35 and −10 regions of the promoters with the bacterial consensus sequence revealed a perfect alignment for the P1 promoter at the −35 position and only two nucleotide changes in the −10 sequence (figure 3b). In addition, the P1 sequence displayed optimal spacing of 17 bp between the −35 and −10 sequence elements. Promoter two contained a near perfect −35 region and an optimal spacer of 17 bp. Although the −10 region is less conserved, there is a TGT motif one base upstream of the −10 region which is known to enhance transcription [17], [18].


Bacterial antigen expression is an important component in inducing an immune response to orally administered Salmonella-delivered DNA vaccines.

Gahan ME, Webster DE, Wesselingh SL, Strugnell RA, Yang J - PLoS ONE (2009)

Location of S. typhimurium promoter sequences within the CMV promoter.(A) DNA sequence of pcDNA3/Cfrag from the start of the CMV promoter highlighting the location of the two primary bacterial promoters (blue), the regulatory regions of the CMV promoter (red) and the start codon of the C-fragment gene (underlined). (B) Alignment of the −35 and −10 promoter regions of the two primary S. typhimurium promoters with the bacterial promoter consensus sequence. The TGT enhancer region located within promoter 2 is shown in bold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698147&req=5

pone-0006062-g003: Location of S. typhimurium promoter sequences within the CMV promoter.(A) DNA sequence of pcDNA3/Cfrag from the start of the CMV promoter highlighting the location of the two primary bacterial promoters (blue), the regulatory regions of the CMV promoter (red) and the start codon of the C-fragment gene (underlined). (B) Alignment of the −35 and −10 promoter regions of the two primary S. typhimurium promoters with the bacterial promoter consensus sequence. The TGT enhancer region located within promoter 2 is shown in bold.
Mentions: The transcription initiation sites of the bacterial promoter sequences were identified by primer extension analysis using RNA extracted from BRD509 (pcDNA3/Cfrag) and BRD509. Two strong primary bacterial transcription initiation sites situated within the CMV promoter and multiple minor promoters were identified (figure 2). The first of these (promoter one, P1) is located 428 base pairs (bp) upstream of the AP1 region of the CMV promoter and the second promoter (promoter two, P2) is 292 bp upstream of AP1. Both bacterial promoters are located upstream of the CMV promoter regulatory elements and the C-fragment start codon (figure 3a). Comparison of the −35 and −10 regions of the promoters with the bacterial consensus sequence revealed a perfect alignment for the P1 promoter at the −35 position and only two nucleotide changes in the −10 sequence (figure 3b). In addition, the P1 sequence displayed optimal spacing of 17 bp between the −35 and −10 sequence elements. Promoter two contained a near perfect −35 region and an optimal spacer of 17 bp. Although the −10 region is less conserved, there is a TGT motif one base upstream of the −10 region which is known to enhance transcription [17], [18].

Bottom Line: Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology.The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter.These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful oral delivery of C-fragment DNA vaccines containing the CMV promoter by S. typhimurium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. michelle.gahan@canberra.edu.au

ABSTRACT

Background: The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contributions of prokaryotic and eukaryotic expression in production of the vaccine antigen. Gene expression in DNA vaccines is commonly under the control of the eukaryotic cytomegalovirus (CMV) promoter. The aim of this study was to identify and disable putative bacterial promoters within the CMV promoter and evaluate the immunogenicity of the resulting DNA vaccine delivered orally by S. typhimurium.

Methodology/principal findings: The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter. These promoters have homology to the bacterial consensus sequence and functional activity. To disable prokaryotic expression from the CMV promoter a series of genetic manipulations were performed to remove the two major bacterial promoters and add a bacteria transcription terminator downstream of the CMV promoter. S. typhimurium was used to immunise BALB/c mice orally with a DNA vaccine encoding the C-fragment of tetanus toxin (TT) under control of the original or the modified CMV promoter. Although both promoters functioned equally well in eukaryotic cells, as indicated by equivalent immune responses following intramuscular delivery, only the original CMV promoter was able to induce an anti-TT specific response following oral delivery by S. typhimurium.

Conclusions: These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful oral delivery of C-fragment DNA vaccines containing the CMV promoter by S. typhimurium.

Show MeSH
Related in: MedlinePlus