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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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Bacterial promoter activity of the original and mutated CMV promoter.(A) Western immunoblots showing expression of C-fragment from S. typhimurium BRD509 containing pcDNA3/Cfrag (CMV), pcDNA3/Cfrag-P1 (P1), pcDNA3/Cfrag-P2 (P2), or pcDNA3/Cfrag-P1/2 (P1/2). Purified C-fragment (pos) and BRD509 alone (neg) were included as controls. (B) To quantify promoter activity, the unmodified pCMV (CMV), pCMV-P1 (P1), pCMV-P2 (P2) and pCMV-P1/2 (P1/2) were cloned into the β-galactosidase vector and promoter activity expressed as units of β-galactosidase activity. Activity of the empty vector (neg) is included as a negative control.
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pone-0006062-g004: Bacterial promoter activity of the original and mutated CMV promoter.(A) Western immunoblots showing expression of C-fragment from S. typhimurium BRD509 containing pcDNA3/Cfrag (CMV), pcDNA3/Cfrag-P1 (P1), pcDNA3/Cfrag-P2 (P2), or pcDNA3/Cfrag-P1/2 (P1/2). Purified C-fragment (pos) and BRD509 alone (neg) were included as controls. (B) To quantify promoter activity, the unmodified pCMV (CMV), pCMV-P1 (P1), pCMV-P2 (P2) and pCMV-P1/2 (P1/2) were cloned into the β-galactosidase vector and promoter activity expressed as units of β-galactosidase activity. Activity of the empty vector (neg) is included as a negative control.

Mentions: C-fragment plasmids were constructed using PCR and restriction enzyme digests with either P1 (pcDNA3/Cfrag-P1), P2 (pcDNA3/Cfrag-P2) or both bacterial promoter regions removed (pcDNA3/Cfrag-P1/2). To determine if bacterial C-fragment expression had been eliminated, total protein from overnight cultures of S. typhimurium BRD509 was analysed by western blot. C-fragment protein expression was detected for BRD509 containing pcDNA3/Cfrag, pcDNA3/Cfrag-P1, pcDNA3/Cfrag-P2 and pcDNA3/Cfrag-P1/2 but not for BRD509 alone (figure 4a). The degree of protein expression for the three modified constructs appeared to be similar to pcDNA3/Cfrag which contains the unmodified CMV promoter.


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)

Bacterial promoter activity of the original and mutated CMV promoter.(A) Western immunoblots showing expression of C-fragment from S. typhimurium BRD509 containing pcDNA3/Cfrag (CMV), pcDNA3/Cfrag-P1 (P1), pcDNA3/Cfrag-P2 (P2), or pcDNA3/Cfrag-P1/2 (P1/2). Purified C-fragment (pos) and BRD509 alone (neg) were included as controls. (B) To quantify promoter activity, the unmodified pCMV (CMV), pCMV-P1 (P1), pCMV-P2 (P2) and pCMV-P1/2 (P1/2) were cloned into the β-galactosidase vector and promoter activity expressed as units of β-galactosidase activity. Activity of the empty vector (neg) is included as a negative control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698147&req=5

pone-0006062-g004: Bacterial promoter activity of the original and mutated CMV promoter.(A) Western immunoblots showing expression of C-fragment from S. typhimurium BRD509 containing pcDNA3/Cfrag (CMV), pcDNA3/Cfrag-P1 (P1), pcDNA3/Cfrag-P2 (P2), or pcDNA3/Cfrag-P1/2 (P1/2). Purified C-fragment (pos) and BRD509 alone (neg) were included as controls. (B) To quantify promoter activity, the unmodified pCMV (CMV), pCMV-P1 (P1), pCMV-P2 (P2) and pCMV-P1/2 (P1/2) were cloned into the β-galactosidase vector and promoter activity expressed as units of β-galactosidase activity. Activity of the empty vector (neg) is included as a negative control.
Mentions: C-fragment plasmids were constructed using PCR and restriction enzyme digests with either P1 (pcDNA3/Cfrag-P1), P2 (pcDNA3/Cfrag-P2) or both bacterial promoter regions removed (pcDNA3/Cfrag-P1/2). To determine if bacterial C-fragment expression had been eliminated, total protein from overnight cultures of S. typhimurium BRD509 was analysed by western blot. C-fragment protein expression was detected for BRD509 containing pcDNA3/Cfrag, pcDNA3/Cfrag-P1, pcDNA3/Cfrag-P2 and pcDNA3/Cfrag-P1/2 but not for BRD509 alone (figure 4a). The degree of protein expression for the three modified constructs appeared to be similar to pcDNA3/Cfrag which contains the unmodified CMV promoter.

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