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1.  The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes▿ †  
Infection and Immunity  2009;77(11):4714-4723.
Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.
doi:10.1128/IAI.00749-09
PMCID: PMC2772542  PMID: 19720758
2.  Sequential B-Cell Epitopes of Bacillus anthracis Lethal Factor Bind Lethal Toxin-Neutralizing Antibodies▿  
Infection and Immunity  2008;77(1):162-169.
The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.
doi:10.1128/IAI.00788-08
PMCID: PMC2612257  PMID: 18981257

Results 1-2 (2)