The detection of antibody to Bacillus anthracis recombinant PA in vaccinated animal serum

Anthrax is a worldwide zoonosis in animals and human. In Mongolia, the confirmed case of anthrax outbreak is reported every year over the past decade. The prevention and control measure of animal anthrax is vaccination using spore of attenuated Sterne strain, but horse does not get vaccinated in Mongolia. In this study, we constructed the recombinant plasmid for over expression of anthrax protective antigen (PA)/GST fusion protein in pGEX-6P-1 vector and purified the recombinant PA (r-PA) using glutathione Sepharose column under native and denaturing conditions. Since both forms of r-PA were recognized by specific antibody against PA, ELISA system to detect antibody titer in vaccinated bovine serum was constructed. Total of 890 vaccinated cattle serum were collected from 178 cattle at 0, 3, 5, 8 and 12 months’ post vaccination. As negative control, 200 cattle serum from Umnugovi aimag were selected which does not have anthrax foci. All serum was tested by rPA indirect ELISA and, antibody to PA were detected in vaccinated cattle serum but were not detected in negative serum. Therefore, rPA should be used in as monitoring of the anthrax vaccination.

Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

Anthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ϕ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ϕ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

Different mechanisms of two anti-anthrax protective antigen antibodies and function comparison between them

Background Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. Methods The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. Results In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. Conclusions Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.

Accurate and selective quantification of anthrax protective antigen in plasma by immunocapture and isotope dilution mass spectrometry

The impact of anthrax PA levels during anthrax infections can be assessed by a novel Ab-capture, tryptic digest LC-MS/MS method.