The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 ؋ 10 8 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ؎ 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296-400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE(306-314) and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE(296-400) in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE(306-314) synthetic peptide and this reaction was inhibited by DE(296-400). Moreover, DE(306-314) could block dengue virus infection of target cells in an in vitro assay. A three-dimensional model of DE revealed that the three amino acids shared by DE(296-400) and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE(306-314) region. Since 4E11 blocked the binding of DE(296-400) to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE(306-314) with target cell-displayed HSHS. Our data suggest that the DE(306-314) segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE(306-314) to HSHS may be antiviral reagents of therapeutic interest.

Background: Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.

Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA 83 ). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA 83 to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (10 7 clones). We isolated a high-affinity ( Bacillus anthracis, the causative agent of anthrax, produces a toxin related to the classic A-B family, which plays a major role in disease pathogenesis. The 83-kDa protective antigen (PA 83 ) is the common cell-binding domain (38) that, after proteolytic activation, can interact with two enzymatically active domains that elicit cell damage, the edema factor (EF; 89 kDa) and lethal factor (LF; 90 kDa). LF is a metalloproteinase specific for mitogen-activated protein kinase kinases. EF is a calmodulin-dependent adenylate cyclase that causes dramatic increases in the intracellular concentration of cAMP. These proteins are secreted by the bacterium as nontoxic monomers and assemble on the surface of receptor-bearing cells to form toxic complexes. After binding to its cellular receptor (4,30,44), PA 83 is cleaved by a furin-like cellular protease(s) (34), causing the release of an amino-terminal 20-kDa fragment and leaving the carboxy-terminal 63-kDa moiety (PA 63 ) bound to its receptor. PA 63 then spontaneously oligomerizes to form a ringshaped homoheptamer that binds LF and/or EF (38) to form the lethal toxin and/or the edema toxin, respectively. Heptamers are internalized by acidic endosomes, and then EF and LF are translocated into the cytosol (32).This pathogenesis and the prophylactic/therapeutic approaches used to treat anthrax are subjects of great interest due to concerns over intentional or inadvertent exposure to aerosols of Bacillus anthracis spores (47