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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

Immune response in mice immunized with IgM-D4/SV40 DNA vaccine.(A) Antibody analyses of IgG subclass after immunization with IgM-D4/SV40 DNA vaccine using EP delivery. The level of IgG subclass was measured by ELISA (1:100 dilutions). (B) DNA vaccination induces IFN-γ responses. Splenocytes from immunized mice were collected 8 weeks after first immunization, cultured in the presence of PA-D4 protein, and IFN-γ level measured by ELISPOT. Values are means ± standard deviations. P<0.05 compared to control.
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pone.0139671.g005: Immune response in mice immunized with IgM-D4/SV40 DNA vaccine.(A) Antibody analyses of IgG subclass after immunization with IgM-D4/SV40 DNA vaccine using EP delivery. The level of IgG subclass was measured by ELISA (1:100 dilutions). (B) DNA vaccination induces IFN-γ responses. Splenocytes from immunized mice were collected 8 weeks after first immunization, cultured in the presence of PA-D4 protein, and IFN-γ level measured by ELISPOT. Values are means ± standard deviations. P<0.05 compared to control.

Mentions: In order to further characterize the immune response stimulated by IgM-D4/SV40, levels of the IgG subtypes were assessed. Mice had predominated production of the Th1-type with IgG2a and IgG2b subclasses, although low levels of IgG1 were also observed (Fig 5A). These results indicate that vaccination with IgM-D4/SV40 induces PA-D4-specific cell-mediated immune responses dominated by a Th1-type of reaction. In addition, increased IgA levels indicated that the IgM-D4/SV40 vaccine also induced the mucosal immune responses.


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)

Immune response in mice immunized with IgM-D4/SV40 DNA vaccine.(A) Antibody analyses of IgG subclass after immunization with IgM-D4/SV40 DNA vaccine using EP delivery. The level of IgG subclass was measured by ELISA (1:100 dilutions). (B) DNA vaccination induces IFN-γ responses. Splenocytes from immunized mice were collected 8 weeks after first immunization, cultured in the presence of PA-D4 protein, and IFN-γ level measured by ELISPOT. Values are means ± standard deviations. P<0.05 compared to control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139671.g005: Immune response in mice immunized with IgM-D4/SV40 DNA vaccine.(A) Antibody analyses of IgG subclass after immunization with IgM-D4/SV40 DNA vaccine using EP delivery. The level of IgG subclass was measured by ELISA (1:100 dilutions). (B) DNA vaccination induces IFN-γ responses. Splenocytes from immunized mice were collected 8 weeks after first immunization, cultured in the presence of PA-D4 protein, and IFN-γ level measured by ELISPOT. Values are means ± standard deviations. P<0.05 compared to control.
Mentions: In order to further characterize the immune response stimulated by IgM-D4/SV40, levels of the IgG subtypes were assessed. Mice had predominated production of the Th1-type with IgG2a and IgG2b subclasses, although low levels of IgG1 were also observed (Fig 5A). These results indicate that vaccination with IgM-D4/SV40 induces PA-D4-specific cell-mediated immune responses dominated by a Th1-type of reaction. In addition, increased IgA levels indicated that the IgM-D4/SV40 vaccine also induced the mucosal immune responses.

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