Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis.
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 50xLD50 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.
Rhie
Gi-eun
GE
Department of Microbiology, National Institute of Health, 194 Tongil-Lo, Seoul 122-701, Republic of Korea. gerhie@nih.go.kr
Park
Young-Mia
YM
Han
Ji-Sun
JS
Yu
Jae-Yon
JY
Seong
Won-Keun
WK
Oh
Hee-Bok
HB
D000595:Amino Acid Sequence; D000818:Animals; D022122:Anthrax Vaccines / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D001407:Bacillus / Q000235:genetics; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002918:Chymotrypsin; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D005798:Genes, Bacterial; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D009154:Mutation; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D017384:Sequence Deletion; D014357:Trypsin
D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C030325:anthrax toxin; D002918:Chymotrypsin; D014357:Trypsin