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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

PA-D4-specific antibody responses in mice immunized with IgM-D4 and IgM-D4/SV40 plasmid.(A) Long-term persistence of PA-D4-specific antibodies induced by DNA vaccine. The serum samples from individual mice immunized with plasmid DNA vaccine were collected at various times and the specific anti-PA-D4 antibody responses were analyzed by ELISA using a single dilution (1:100). Mice were immunized three times (week 0, 2, 4) with plasmids as indicated. (B) Induction kinetics of PA-D4 specific antibodies. Serial dilutions of pooled sera from mice were tested in a PA-D4-specific ELISA to determine the kinetics of PA-specific induction. Values are means ± standard deviations.
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pone.0139671.g003: PA-D4-specific antibody responses in mice immunized with IgM-D4 and IgM-D4/SV40 plasmid.(A) Long-term persistence of PA-D4-specific antibodies induced by DNA vaccine. The serum samples from individual mice immunized with plasmid DNA vaccine were collected at various times and the specific anti-PA-D4 antibody responses were analyzed by ELISA using a single dilution (1:100). Mice were immunized three times (week 0, 2, 4) with plasmids as indicated. (B) Induction kinetics of PA-D4 specific antibodies. Serial dilutions of pooled sera from mice were tested in a PA-D4-specific ELISA to determine the kinetics of PA-specific induction. Values are means ± standard deviations.

Mentions: To assess the immunogenicity responses induced by plasmid DNA immunization, BALB/c mice (n = 5–7) were immunized with various DNA constructs by IM injection. Mice were vaccinated 3 times at 2-week intervals for 44 weeks with one of the following plasmids: IgM-D4, IgM-D4/SV40, pcDNA/SV40, or pcDNA 3.1 vector only as a control. Blood was collected at various times following immunization for determination of anti-PA-D4 IgG titers in serum. As shown in Fig 3A, IgG anti-PA-D4 antibodies were induced at 5 weeks after the first immunization with IgM-D4/SV40 and remained elevated for the 44-week duration of the study. The PA-D4-specific IgG were also induced in mice immunized with IgM-D4; however, the titers were lower, by approximately 10 times less than mice vaccinated with IgM-D4/SV40 (Fig 3B). Our results clearly demonstrated that SV40 enhancer is able to significantly increase the rate of antibody production.


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)

PA-D4-specific antibody responses in mice immunized with IgM-D4 and IgM-D4/SV40 plasmid.(A) Long-term persistence of PA-D4-specific antibodies induced by DNA vaccine. The serum samples from individual mice immunized with plasmid DNA vaccine were collected at various times and the specific anti-PA-D4 antibody responses were analyzed by ELISA using a single dilution (1:100). Mice were immunized three times (week 0, 2, 4) with plasmids as indicated. (B) Induction kinetics of PA-D4 specific antibodies. Serial dilutions of pooled sera from mice were tested in a PA-D4-specific ELISA to determine the kinetics of PA-specific induction. Values are means ± standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139671.g003: PA-D4-specific antibody responses in mice immunized with IgM-D4 and IgM-D4/SV40 plasmid.(A) Long-term persistence of PA-D4-specific antibodies induced by DNA vaccine. The serum samples from individual mice immunized with plasmid DNA vaccine were collected at various times and the specific anti-PA-D4 antibody responses were analyzed by ELISA using a single dilution (1:100). Mice were immunized three times (week 0, 2, 4) with plasmids as indicated. (B) Induction kinetics of PA-D4 specific antibodies. Serial dilutions of pooled sera from mice were tested in a PA-D4-specific ELISA to determine the kinetics of PA-specific induction. Values are means ± standard deviations.
Mentions: To assess the immunogenicity responses induced by plasmid DNA immunization, BALB/c mice (n = 5–7) were immunized with various DNA constructs by IM injection. Mice were vaccinated 3 times at 2-week intervals for 44 weeks with one of the following plasmids: IgM-D4, IgM-D4/SV40, pcDNA/SV40, or pcDNA 3.1 vector only as a control. Blood was collected at various times following immunization for determination of anti-PA-D4 IgG titers in serum. As shown in Fig 3A, IgG anti-PA-D4 antibodies were induced at 5 weeks after the first immunization with IgM-D4/SV40 and remained elevated for the 44-week duration of the study. The PA-D4-specific IgG were also induced in mice immunized with IgM-D4; however, the titers were lower, by approximately 10 times less than mice vaccinated with IgM-D4/SV40 (Fig 3B). Our results clearly demonstrated that SV40 enhancer is able to significantly increase the rate of antibody production.

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