RESUMO
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.
Assuntos
Antígenos de Bactérias/genética , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Toxinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Vacinas contra Antraz/genética , Vacinas contra Antraz/imunologia , Vacinas contra Antraz/isolamento & purificação , Vacinas contra Antraz/toxicidade , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/toxicidade , Bacillus/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Quimotripsina , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Cobaias , Humanos , Camundongos , Mutação , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Deleção de Sequência , TripsinaRESUMO
We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.