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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

DNA dose effect on kinetics of PA-D4-specific antibody response elicited by EP or conventional injection.(A) Antibody levels are expressed as endpoint titers obtained using the ELISA assay. IgM-D4/SV40 DNA at the indicated doses was injected into mice at 0, 2 and 4 wk, and sera were collected at 8 wk. Symbols show individual values; the bars represent mean titer for each group. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests. (B) Antibody response following various doses of IgM-D4/SV40 DNA vaccine administration via EP. Mice were immunized three times with plasmids as indicated. Antibody levels are expressed as OD450 values after the serum samples were diluted 100-fold.
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pone.0139671.g004: DNA dose effect on kinetics of PA-D4-specific antibody response elicited by EP or conventional injection.(A) Antibody levels are expressed as endpoint titers obtained using the ELISA assay. IgM-D4/SV40 DNA at the indicated doses was injected into mice at 0, 2 and 4 wk, and sera were collected at 8 wk. Symbols show individual values; the bars represent mean titer for each group. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests. (B) Antibody response following various doses of IgM-D4/SV40 DNA vaccine administration via EP. Mice were immunized three times with plasmids as indicated. Antibody levels are expressed as OD450 values after the serum samples were diluted 100-fold.

Mentions: To enhance antibody responses to IgM-D4/SV40, DNA dose effect on the level of anti-PA-D4 IgG production was assessed. As shown in Fig 4A, when a dose of 200 μg of DNA was administered by conventional needle injection, the antibody titer was higher than when 100 μg of DNA was administered by conventional needle injection. When 50 μg of DNA was delivered by electroporation (EP), antibody responses in mice were rapidly and consistently induced (Fig 4B) and remained significantly higher than the responses to 200 μg of DNA administered by conventional needle injection. More importantly, even the lowest DNA dose (10 μg) delivered by EP resulted in the induction of similar levels of anti-PA-D4 IgG titers compared with those elicited by a 10-fold higher dose of DNA administered by conventional needle injection. Thus, EP-based delivery dramatically enhanced the potency of the IgM-D4/SV40 DNA vaccine.


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)

DNA dose effect on kinetics of PA-D4-specific antibody response elicited by EP or conventional injection.(A) Antibody levels are expressed as endpoint titers obtained using the ELISA assay. IgM-D4/SV40 DNA at the indicated doses was injected into mice at 0, 2 and 4 wk, and sera were collected at 8 wk. Symbols show individual values; the bars represent mean titer for each group. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests. (B) Antibody response following various doses of IgM-D4/SV40 DNA vaccine administration via EP. Mice were immunized three times with plasmids as indicated. Antibody levels are expressed as OD450 values after the serum samples were diluted 100-fold.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139671.g004: DNA dose effect on kinetics of PA-D4-specific antibody response elicited by EP or conventional injection.(A) Antibody levels are expressed as endpoint titers obtained using the ELISA assay. IgM-D4/SV40 DNA at the indicated doses was injected into mice at 0, 2 and 4 wk, and sera were collected at 8 wk. Symbols show individual values; the bars represent mean titer for each group. P values were determined using one-way ANOVA followed by Tukey multiple comparison tests. (B) Antibody response following various doses of IgM-D4/SV40 DNA vaccine administration via EP. Mice were immunized three times with plasmids as indicated. Antibody levels are expressed as OD450 values after the serum samples were diluted 100-fold.
Mentions: To enhance antibody responses to IgM-D4/SV40, DNA dose effect on the level of anti-PA-D4 IgG production was assessed. As shown in Fig 4A, when a dose of 200 μg of DNA was administered by conventional needle injection, the antibody titer was higher than when 100 μg of DNA was administered by conventional needle injection. When 50 μg of DNA was delivered by electroporation (EP), antibody responses in mice were rapidly and consistently induced (Fig 4B) and remained significantly higher than the responses to 200 μg of DNA administered by conventional needle injection. More importantly, even the lowest DNA dose (10 μg) delivered by EP resulted in the induction of similar levels of anti-PA-D4 IgG titers compared with those elicited by a 10-fold higher dose of DNA administered by conventional needle injection. Thus, EP-based delivery dramatically enhanced the potency of the IgM-D4/SV40 DNA vaccine.

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