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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

Secreted PA-D4 proteins present in cell culture supernatants were analyzed with capture ELISA.The 293T cells were transfected with IgM-D4, IgK-D4, or IgM-D4/SV40 plasmids. At 48 h after transfection, cell culture supernatants were collected and assayed for secreted PA-D4 proteins with capture ELISA. Values are means ± standard deviations. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests.
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pone.0139671.g002: Secreted PA-D4 proteins present in cell culture supernatants were analyzed with capture ELISA.The 293T cells were transfected with IgM-D4, IgK-D4, or IgM-D4/SV40 plasmids. At 48 h after transfection, cell culture supernatants were collected and assayed for secreted PA-D4 proteins with capture ELISA. Values are means ± standard deviations. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests.

Mentions: To determine if PA-D4 could be stably expressed in and secreted from mammalian cells, plasmid IgM-D4 or IgK-D4 was transfected into 293T cells. A comparison of PA-D4 expressed in the culture medium by IgM-D4 and IgK-D4 was made using a capture ELISA. The measurement of expressed PA-D4 by a capture ELISA showed that the level of PA-D4 secreted into the culture supernatant was higher for IgM-D4 than for IgK-D4. As shown in Fig 2, IgM-D4 contained the mouse IgM secretion signal, and 4-fold-more PA-D4 protein was detected in the IgM-D4 cell culture supernatant 48 h post-transfection than in the supernatant from the IgK-D4 cells, which contained a mouse IgK signal.


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)

Secreted PA-D4 proteins present in cell culture supernatants were analyzed with capture ELISA.The 293T cells were transfected with IgM-D4, IgK-D4, or IgM-D4/SV40 plasmids. At 48 h after transfection, cell culture supernatants were collected and assayed for secreted PA-D4 proteins with capture ELISA. Values are means ± standard deviations. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139671.g002: Secreted PA-D4 proteins present in cell culture supernatants were analyzed with capture ELISA.The 293T cells were transfected with IgM-D4, IgK-D4, or IgM-D4/SV40 plasmids. At 48 h after transfection, cell culture supernatants were collected and assayed for secreted PA-D4 proteins with capture ELISA. Values are means ± standard deviations. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests.
Mentions: To determine if PA-D4 could be stably expressed in and secreted from mammalian cells, plasmid IgM-D4 or IgK-D4 was transfected into 293T cells. A comparison of PA-D4 expressed in the culture medium by IgM-D4 and IgK-D4 was made using a capture ELISA. The measurement of expressed PA-D4 by a capture ELISA showed that the level of PA-D4 secreted into the culture supernatant was higher for IgM-D4 than for IgK-D4. As shown in Fig 2, IgM-D4 contained the mouse IgM secretion signal, and 4-fold-more PA-D4 protein was detected in the IgM-D4 cell culture supernatant 48 h post-transfection than in the supernatant from the IgK-D4 cells, which contained a mouse IgK signal.

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