Laffly Emmanuelle, Danjou Ludivine, Condemine Florence, Vidal Dominique, Drouet Emmanuel, Lefranc Marie-Paule, Bottex Chantal, Thullier Philippe
Immunobiologie, Département de biologie des agents transmissibles, Centre de Recherche du Service de Santé des Armées, La Tronche 38702, France.
Antimicrob Agents Chemother. 2005 Aug;49(8):3414-20. doi: 10.1128/AAC.49.8.3414-3420.2005.
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 (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.
正如最近的生物恐怖袭击所凸显的那样，人类炭疽感染并非总能通过抗生素得到成功治疗；因此，未来显然需要辅助疗法。尤其需要进一步开发能够中和分泌毒素的辅助疗法，例如针对83 kDa保护性抗原（PA(83)）的抗体。在缺乏人类供体的情况下，我们用PA(83)免疫了一只猕猴（食蟹猴），以获得适合作为人类炭疽感染辅助疗法的此类抗体。通过使用骨髓作为模板，我们通过PCR扩增了特定的Fab编码基因，并将其克隆为一个免疫文库（10⁷个克隆）。我们通过淘选从该文库中分离出一种高亲和力（平衡解离常数[K(D)]，3.4 nM）、高度中和（50%抑制浓度，5.6±0.13 nM）的Fab（命名为35PA(83)）。通过Pepscan分析确定其表位位于PA(83)的686至694位残基之间，并且是直接与细胞受体相互作用区域的一部分。因此，35PA(83)可能通过直接竞争其受体来中和炭疽毒素。编码35PA(83)的基因与人类免疫球蛋白种系的基因相似，并且通过IMGT/V-QUEST分析被归入人类V、(D)或J基因的亚组。35PA(83)的框架区域与每个亚组的代表性等位基因有92%的同一性。与相关人类种系基因编码的框架区域相比，35PA(83)分析的74个（VH）或75个（VK）氨基酸中只有2个具有不同的化学特性。与人类框架区域的高度同一性使得35PA(83)非常适合作为完整的灵长类化免疫球蛋白G进行表达，并证明了在没有免疫的人类浆细胞供体时使用猕猴Fab的实用性。
