Recombinant Bacillus anthracis Lethal Factor

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B, anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LP to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current Live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.

Monoclonal antibodies (MAbs) are potential therapeutic agents against Bacillus anthracis toxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in an in vitro mouse macrophage system but did not provide significant protection in vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection both in vitro and in vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralization in vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection with B. anthracis strain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.