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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

Biodistribution of DNA vaccines.Mice were immunized with 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA and plasmid copy numbers were measured with quantitative real-time polymerase chain reaction assay at various points after IM injection. PCR reaction products were not detected for control animals (vector only). Data are expressed as PCN per microgram of DNA. Values are means ± standard deviations.
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pone.0139671.g006: Biodistribution of DNA vaccines.Mice were immunized with 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA and plasmid copy numbers were measured with quantitative real-time polymerase chain reaction assay at various points after IM injection. PCR reaction products were not detected for control animals (vector only). Data are expressed as PCN per microgram of DNA. Values are means ± standard deviations.

Mentions: To analyze the distribution profiles of the IgM-D4/SV40 DNA vaccine, we used qPCR to quantify the level of DNA vaccine in various tissues. In a mouse time course biodistribution study, 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA were administered intramuscularly and pharmacokinetic analysis of the plasmid DNA in the tissues was performed. The distribution of plasmid DNA in the tissues was measured from 1 h to 48 h after injection. The total DNA was extracted from the lung, liver, spleen, heart, brain, kidney, blood, and muscle at the different time points. Plasmid DNA copies were detectable in all tissues 48 h post-administration, although levels of plasmid DNA copies differed between tissues (Fig 6). One hour after injection of plasmid DNA, the highest copy numbers were observed in the injected muscles. However, 3 h post-administration, the copy numbers rapidly decreased. At 48 h after administration of plasmid DNA, plasmid copies remaining in the muscle decreased 1.5 × 106-fold relative to the levels at 1 h. The level of plasmids in most of the organs increased substantially over time. The plasmid level in the spleen increased 105-fold by 48 h post-administration compared with the early time point (1 h). The plasmid levels in organs were detected at 48 h in the following order: spleen, lung, heart, kidney, liver, and brain. However, the blood showed a different biodistribution profile in which the plasmid copies peaked at 24 h post-administration and rapidly declined thereafter.


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)

Biodistribution of DNA vaccines.Mice were immunized with 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA and plasmid copy numbers were measured with quantitative real-time polymerase chain reaction assay at various points after IM injection. PCR reaction products were not detected for control animals (vector only). Data are expressed as PCN per microgram of DNA. Values are means ± standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139671.g006: Biodistribution of DNA vaccines.Mice were immunized with 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA and plasmid copy numbers were measured with quantitative real-time polymerase chain reaction assay at various points after IM injection. PCR reaction products were not detected for control animals (vector only). Data are expressed as PCN per microgram of DNA. Values are means ± standard deviations.
Mentions: To analyze the distribution profiles of the IgM-D4/SV40 DNA vaccine, we used qPCR to quantify the level of DNA vaccine in various tissues. In a mouse time course biodistribution study, 3.5 × 1013 copies IgM-D4/SV40 plasmid DNA were administered intramuscularly and pharmacokinetic analysis of the plasmid DNA in the tissues was performed. The distribution of plasmid DNA in the tissues was measured from 1 h to 48 h after injection. The total DNA was extracted from the lung, liver, spleen, heart, brain, kidney, blood, and muscle at the different time points. Plasmid DNA copies were detectable in all tissues 48 h post-administration, although levels of plasmid DNA copies differed between tissues (Fig 6). One hour after injection of plasmid DNA, the highest copy numbers were observed in the injected muscles. However, 3 h post-administration, the copy numbers rapidly decreased. At 48 h after administration of plasmid DNA, plasmid copies remaining in the muscle decreased 1.5 × 106-fold relative to the levels at 1 h. The level of plasmids in most of the organs increased substantially over time. The plasmid level in the spleen increased 105-fold by 48 h post-administration compared with the early time point (1 h). The plasmid levels in organs were detected at 48 h in the following order: spleen, lung, heart, kidney, liver, and brain. However, the blood showed a different biodistribution profile in which the plasmid copies peaked at 24 h post-administration and rapidly declined thereafter.

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