Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Infect Immun. 1998 Oct;66(10):4817-22.

Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate.

Author information

  • 1Division of Toxinology, United States Army Research Institute for Infectious Diseases, Frederick, Maryland 21702-5011, USA.

Abstract

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of </=100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.

PMID:
9746584
[PubMed - indexed for MEDLINE]
PMCID:
PMC108595
Free PMC Article

Images from this publication.See all images (4)Free text

FIG. 1
FIG. 2
FIG. 3
FIG. 4
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk