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Antimicrobial Agents and Chemotherapy, August 2005, p. 3414-3420, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3414-3420.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Emmanuelle Laffly,1 Ludivine Danjou,2 Florence Condemine,2 Dominique Vidal,2 Emmanuel Drouet,1 Marie-Paule Lefranc,3 Chantal Bottex,2 and Philippe Thullier2*

EA 2939 GDR CNRS, Virologie moléculaire et structurale, La Tronche, France,1 Département de biologie des agents transmissibles, Centre de Recherche du Service de Santé des Armées, La Tronche, France,2 Laboratoire d'ImmunoGénétique Moléculaire (LIGM), Université Montpellier II, UPR CNRS 1142, Institut de Génétique Humaine (IGH), Montpellier, France, and Institut Universitaire de France, Paris, France3

Received 28 September 2004/ Returned for modification 18 November 2004/ Accepted 24 May 2005

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 (PA83). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA83 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 (107 clones). We isolated a high-affinity (equilibrium dissociation constant [KD], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 ± 0.13 nM) Fab (designated 35PA83) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA83 and is part of the region which directly interacts with the cell receptor. 35PA83 may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA83 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 35PA83 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 35PA83 have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA83 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.

* Corresponding author. Mailing address: Immunobiologie, Département de biologie des agents transmissibles, Centre de Recherche du Service de Santé des Armées, La Tronche 38702, France. Phone: 33 4 76 63 69 14. Fax: 33 4 76 63 69 17. E-mail: pthullier{at}yahoo.com.

Antimicrobial Agents and Chemotherapy, August 2005, p. 3414-3420, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3414-3420.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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