dThe nontoxic N-terrninal fragment of Bacillus anthracis edema factor (EF) was evaluated as a candidate antigen in an anthrax vaccine using a replication-incompetent adenoviral vector. An E1/E3 deleted adenovirus (Ad/EFn) encoding the N-terminal region 1-254 amino acids of the edema factor (EFn) was constructed using the native DNA sequence of EFn. Intramuscular immunization three times with 108 plaque forming units (pfu)/dose of Ad/EFn in A/J mice resulted in 37% and 57% protection against a subcutaneous challenge with B. anthracis Sterne strain spores at a dosage of 200 x LD50 and 100 x LD50, respectively. EF-specific serum IgG responses (including total IgG, IgG1, and IgG2a isotype titers) were robust in the Ad/EFn immunized animals. Interestingly, anti-EF antibodies cross-reacted with anthrax lethal factor (LF), and had a neutralizing capability against both anthrax lethal toxin (Letx) and edema toxin (Edtx), as demonstrated by in vitro toxin neutralization assays using J774A.1 mouse macrophage and Chinese hamster ovary cell (CHO), respectively. Our data suggest that EF plays a role in eliciting protective immunity against anthrax, and that it should be included in a new generation multi-component subunit vaccine. (c) 2005 Elsevier Ltd. All rights reserved.

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-bindin component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase 1, enolase 1, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death. (C) 2004 Elsevier B.V. All rights reserved.