Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis

• Published in: FEMS immunology and medical microbiology Vol. 45; no. 2; pp. 341 - 347
• Main Authors: Rhie, Gi-eun, Park, Young-Mia, Han, Ji-Sun, Yu, Jae-Yon, Seong, Won-Keun, Oh, Hee-Bok
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01.08.2005; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Alum; Amino Acid Sequence; Ammonium; Ammonium sulfate; Animals; Anion exchange; Anion exchanging; Anthrax; Anthrax vaccine; Anthrax Vaccines - genetics; Anthrax Vaccines - immunology; Anthrax Vaccines - isolation & purification; Anthrax Vaccines - toxicity; Antibodies; Antibodies, Bacterial - biosynthesis; Antigens; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Antigens, Bacterial - isolation & purification; Antigens, Bacterial - toxicity; Applied microbiology; Bacillus - genetics; Bacillus anthracis; Bacillus anthracis - genetics; Bacillus anthracis - immunology; Bacterial Toxins - genetics; Bacterial Toxins - immunology; Bacterial Toxins - isolation & purification; Bacterial Toxins - toxicity; Base Sequence; Binding Sites - genetics; Biological and medical sciences; Cell culture; Cell Line; Chymotrypsin; Clonal deletion; Cytotoxicity; Deletion mutant; DNA, Bacterial - genetics; Female; Fundamental and applied biological sciences. Psychology; Gel filtration; Genes, Bacterial; Guinea Pigs; Humans; Hydrophobicity; Lethal factor; Mice; Microbiology; Mutants; Mutation; Neutralization Tests; Neutralizing; Protective antigen; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - toxicity; Sequence Deletion; Toxicity; Trypsin; Vaccines; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects); Bacillus anthracis; Protective antigen; Anthrax vaccine; Deletion mutant; Microbiology; Vaccine; Bacillaceae; Bacillus anthacis; Infection; Antigen; Immunology; Bacillales; Anthrax; Efficiency; Bacteriosis; Deletion; Bacteria; Mutation
• ISSN: 0928-8244; 1574-695X; 2049-632X
• DOI: 10.1016/j.femsim.2005.05.009

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163–168) and delPA (313–314), that lack trypsin (S 163– R 164–K 165–K 166–R 167 –S 168) or chymotrypsin cleavage sequence ( F 313–F 314 ), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163–168) and delPA (313–314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50 × LD 50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163–168) and delPA (313–314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.