Immunogenicity of CT-B::DTx-B, CT-B::PT-S1*,S2, and CT-B::TT-B chimeric proteins: An approach to develop a safer DPT vaccine

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Abstract

We report here the construction of several chimeric genes encoding the binding domains of diphtheria (DTx-B), pertussis (PT-S2), and tetanus (TT-B) toxins, as well as a modified PT-S1 enzymatic subunit, which were placed downstream and in-frame with the immunomodulatory cholera toxin B-subunit (CT-B) gene. Each chimeric gene construct was hyperexpressed in Escherichia coli, and the fusion proteins, that is, CT-B::DTx-B, CT-B::PT-S2, and CT-B::TT-B, reacted with antibodies to each component of the chimeras in an ELISA and Western blot analysis. The hyperproduced proteins induced significant antibody titers in mice against both components of the chimeric proteins. A mutagenized PT-S1 subunit gene construct was made by first using site-directed mutagenesis to modify codons encoding amino acid residues Arg9 and Glu129, which are responsible for the ADP-ribosyltransferase activity of the PT-S1 subunit. The codons for these amino acids were replaced with those encoding Lys and Gly, respectively, and the genetically inactivated PT-S1(*): gene was ligated downstream of the CT-B gene and expressed in E. coli. After hyperexpression, antibodies generated against the CT-B::PT-S1(*) construct neutralized the Chinese hamster ovary (CHO) cell clustering activity, which is a typical PT biologic response. Further, the antibodies blocked the elongation of CHO cells, which is a characteristic response of these cells to CT. These chimeric antigens may be beneficial in the development of alternative recombinant vaccines with minimal toxic side effects compared with those seen with the whole cell DPT vaccine.

Original languageEnglish (US)
Pages (from-to)1-13
Number of pages13
JournalVaccine Research
Volume6
Issue number1
StatePublished - 1997

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Diphtheria-Tetanus-Pertussis Vaccine
Cholera Toxin
Genes
Antibodies
Cricetulus
Codon
Ovary
ADP Ribose Transferases
Amino Acids
Synthetic Vaccines
Diphtheria
Whooping Cough
Poisons
Escherichia coli Proteins
Tetanus
Site-Directed Mutagenesis
Cluster Analysis
IgA receptor
Proteins
Western Blotting

ASJC Scopus subject areas

  • Immunology
  • Microbiology (medical)

Cite this

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title = "Immunogenicity of CT-B::DTx-B, CT-B::PT-S1*,S2, and CT-B::TT-B chimeric proteins: An approach to develop a safer DPT vaccine",
abstract = "We report here the construction of several chimeric genes encoding the binding domains of diphtheria (DTx-B), pertussis (PT-S2), and tetanus (TT-B) toxins, as well as a modified PT-S1 enzymatic subunit, which were placed downstream and in-frame with the immunomodulatory cholera toxin B-subunit (CT-B) gene. Each chimeric gene construct was hyperexpressed in Escherichia coli, and the fusion proteins, that is, CT-B::DTx-B, CT-B::PT-S2, and CT-B::TT-B, reacted with antibodies to each component of the chimeras in an ELISA and Western blot analysis. The hyperproduced proteins induced significant antibody titers in mice against both components of the chimeric proteins. A mutagenized PT-S1 subunit gene construct was made by first using site-directed mutagenesis to modify codons encoding amino acid residues Arg9 and Glu129, which are responsible for the ADP-ribosyltransferase activity of the PT-S1 subunit. The codons for these amino acids were replaced with those encoding Lys and Gly, respectively, and the genetically inactivated PT-S1(*): gene was ligated downstream of the CT-B gene and expressed in E. coli. After hyperexpression, antibodies generated against the CT-B::PT-S1(*) construct neutralized the Chinese hamster ovary (CHO) cell clustering activity, which is a typical PT biologic response. Further, the antibodies blocked the elongation of CHO cells, which is a characteristic response of these cells to CT. These chimeric antigens may be beneficial in the development of alternative recombinant vaccines with minimal toxic side effects compared with those seen with the whole cell DPT vaccine.",
author = "Y. Lu and Johnny Peterson and Ashok Chopra",
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T1 - Immunogenicity of CT-B::DTx-B, CT-B::PT-S1*,S2, and CT-B::TT-B chimeric proteins: An approach to develop a safer DPT vaccine

AU - Lu, Y.

AU - Peterson, Johnny

AU - Chopra, Ashok

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N2 - We report here the construction of several chimeric genes encoding the binding domains of diphtheria (DTx-B), pertussis (PT-S2), and tetanus (TT-B) toxins, as well as a modified PT-S1 enzymatic subunit, which were placed downstream and in-frame with the immunomodulatory cholera toxin B-subunit (CT-B) gene. Each chimeric gene construct was hyperexpressed in Escherichia coli, and the fusion proteins, that is, CT-B::DTx-B, CT-B::PT-S2, and CT-B::TT-B, reacted with antibodies to each component of the chimeras in an ELISA and Western blot analysis. The hyperproduced proteins induced significant antibody titers in mice against both components of the chimeric proteins. A mutagenized PT-S1 subunit gene construct was made by first using site-directed mutagenesis to modify codons encoding amino acid residues Arg9 and Glu129, which are responsible for the ADP-ribosyltransferase activity of the PT-S1 subunit. The codons for these amino acids were replaced with those encoding Lys and Gly, respectively, and the genetically inactivated PT-S1(*): gene was ligated downstream of the CT-B gene and expressed in E. coli. After hyperexpression, antibodies generated against the CT-B::PT-S1(*) construct neutralized the Chinese hamster ovary (CHO) cell clustering activity, which is a typical PT biologic response. Further, the antibodies blocked the elongation of CHO cells, which is a characteristic response of these cells to CT. These chimeric antigens may be beneficial in the development of alternative recombinant vaccines with minimal toxic side effects compared with those seen with the whole cell DPT vaccine.

AB - We report here the construction of several chimeric genes encoding the binding domains of diphtheria (DTx-B), pertussis (PT-S2), and tetanus (TT-B) toxins, as well as a modified PT-S1 enzymatic subunit, which were placed downstream and in-frame with the immunomodulatory cholera toxin B-subunit (CT-B) gene. Each chimeric gene construct was hyperexpressed in Escherichia coli, and the fusion proteins, that is, CT-B::DTx-B, CT-B::PT-S2, and CT-B::TT-B, reacted with antibodies to each component of the chimeras in an ELISA and Western blot analysis. The hyperproduced proteins induced significant antibody titers in mice against both components of the chimeric proteins. A mutagenized PT-S1 subunit gene construct was made by first using site-directed mutagenesis to modify codons encoding amino acid residues Arg9 and Glu129, which are responsible for the ADP-ribosyltransferase activity of the PT-S1 subunit. The codons for these amino acids were replaced with those encoding Lys and Gly, respectively, and the genetically inactivated PT-S1(*): gene was ligated downstream of the CT-B gene and expressed in E. coli. After hyperexpression, antibodies generated against the CT-B::PT-S1(*) construct neutralized the Chinese hamster ovary (CHO) cell clustering activity, which is a typical PT biologic response. Further, the antibodies blocked the elongation of CHO cells, which is a characteristic response of these cells to CT. These chimeric antigens may be beneficial in the development of alternative recombinant vaccines with minimal toxic side effects compared with those seen with the whole cell DPT vaccine.

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