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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 (equilibrium dissociation constant [K(D)], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 +/- 0.13 nM) Fab (designated 35PA(83)) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA(83) and is part of the region which directly interacts with the cell receptor. 35PA(83) may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA(83) were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA(83) framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA(83) have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA(83) well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

antimicrobial agents and chemotherapy

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of <i>Bacillus anthracis</i> by Binding to the Segment of PA between Residues 686 and 694

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694