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Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge.

Kim NY, Chang DS, Kim Y, Kim CH, Hur GH, Yang JM, Shin S - PLoS ONE (2015)

Bottom Line: The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium.Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer.The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Sogang University, Seoul, Republic of Korea.

ABSTRACT
Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.

No MeSH data available.


Related in: MedlinePlus

Expression of PA-D4 after in vitro transfection.293T cells were transfected with pEGFP/D4 plasmids. At 48 h after transfection, the cells were harvested and assayed for expression of EGFP fusion PA-D4. (A) Confocal fluorescence microscopy to demonstrate the expression PA-D4 protein. Magnification is 200×. (B) Western blot analysis demonstrating the expression of PA-D4. The proteins were separated in SDS-PAGE using a 4–20% polyacrylamide gel before blotting. Expression of PA-D4 protein was detected with mouse polyclonal antibodies against PA-D4. Lane 1, vector only control cell lysates; lane 2, original type transfected cell lysates; lane 3, codon-optimized transfected cell lysates.
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pone.0139671.g001: Expression of PA-D4 after in vitro transfection.293T cells were transfected with pEGFP/D4 plasmids. At 48 h after transfection, the cells were harvested and assayed for expression of EGFP fusion PA-D4. (A) Confocal fluorescence microscopy to demonstrate the expression PA-D4 protein. Magnification is 200×. (B) Western blot analysis demonstrating the expression of PA-D4. The proteins were separated in SDS-PAGE using a 4–20% polyacrylamide gel before blotting. Expression of PA-D4 protein was detected with mouse polyclonal antibodies against PA-D4. Lane 1, vector only control cell lysates; lane 2, original type transfected cell lysates; lane 3, codon-optimized transfected cell lysates.

Mentions: The fourth domain of PA has been shown to mediate binding of PA to its cellular receptor for the anthrax toxin and thus, PA-D4 is a particularly attractive target antigen for the DNA vaccine against anthrax [2]. Therefore, these experiments were carried out using the gene encoding PA-D4, which is a target antigen for the DNA vaccine. A number of studies have found that there is a good correlation between the codon bias of a gene and its level of expression [28,29]. Therefore, we designed a codon-optimized synthetic gene encoding PA-D4, that with most of the rare codons replaced by codons that are most frequently used in human cells. To assess the transfection efficiency of the codon-optimized PA-D4 gene, we decided to construct a eukaryotic expression vector, pEGFP/D4, encoding a PA-D4 with pEGFP-C1 as the backbone. The plasmid vector encoding PA-D4 was transfected into 293T cells. GFP expression was evaluated by fluorescence microscopy 48 h after transfection. As shown in Fig 1A, fluorescence was visualized in cells transfected with codon-optimized PA-D4 but not in original PA-D4 transfected cells. Fluorescence indicates the production of full-length EGFP-D4 fusion protein, since the EGFP component is at the amino terminus.


Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge.

Kim NY, Chang DS, Kim Y, Kim CH, Hur GH, Yang JM, Shin S - PLoS ONE (2015)

Expression of PA-D4 after in vitro transfection.293T cells were transfected with pEGFP/D4 plasmids. At 48 h after transfection, the cells were harvested and assayed for expression of EGFP fusion PA-D4. (A) Confocal fluorescence microscopy to demonstrate the expression PA-D4 protein. Magnification is 200×. (B) Western blot analysis demonstrating the expression of PA-D4. The proteins were separated in SDS-PAGE using a 4–20% polyacrylamide gel before blotting. Expression of PA-D4 protein was detected with mouse polyclonal antibodies against PA-D4. Lane 1, vector only control cell lysates; lane 2, original type transfected cell lysates; lane 3, codon-optimized transfected cell lysates.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4591996&req=5

pone.0139671.g001: Expression of PA-D4 after in vitro transfection.293T cells were transfected with pEGFP/D4 plasmids. At 48 h after transfection, the cells were harvested and assayed for expression of EGFP fusion PA-D4. (A) Confocal fluorescence microscopy to demonstrate the expression PA-D4 protein. Magnification is 200×. (B) Western blot analysis demonstrating the expression of PA-D4. The proteins were separated in SDS-PAGE using a 4–20% polyacrylamide gel before blotting. Expression of PA-D4 protein was detected with mouse polyclonal antibodies against PA-D4. Lane 1, vector only control cell lysates; lane 2, original type transfected cell lysates; lane 3, codon-optimized transfected cell lysates.
Mentions: The fourth domain of PA has been shown to mediate binding of PA to its cellular receptor for the anthrax toxin and thus, PA-D4 is a particularly attractive target antigen for the DNA vaccine against anthrax [2]. Therefore, these experiments were carried out using the gene encoding PA-D4, which is a target antigen for the DNA vaccine. A number of studies have found that there is a good correlation between the codon bias of a gene and its level of expression [28,29]. Therefore, we designed a codon-optimized synthetic gene encoding PA-D4, that with most of the rare codons replaced by codons that are most frequently used in human cells. To assess the transfection efficiency of the codon-optimized PA-D4 gene, we decided to construct a eukaryotic expression vector, pEGFP/D4, encoding a PA-D4 with pEGFP-C1 as the backbone. The plasmid vector encoding PA-D4 was transfected into 293T cells. GFP expression was evaluated by fluorescence microscopy 48 h after transfection. As shown in Fig 1A, fluorescence was visualized in cells transfected with codon-optimized PA-D4 but not in original PA-D4 transfected cells. Fluorescence indicates the production of full-length EGFP-D4 fusion protein, since the EGFP component is at the amino terminus.

Bottom Line: The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium.Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer.The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Sogang University, Seoul, Republic of Korea.

ABSTRACT
Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.

No MeSH data available.


Related in: MedlinePlus