Vuong Christine N, Chou Wen-Ko, Kuttappan Vivek A, Hargis Billy M, Bielke Lisa R, Berghman Luc R
Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, United States.
Department of Poultry Science, Texas A&M University, College Station, TX, United States.
Front Vet Sci. 2017 Jun 13;4:91. doi: 10.3389/fvets.2017.00091. eCollection 2017.
targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC) enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin's hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and identification of neutralizing epitopes in microbial toxins and enzymes within 2 weeks from analysis of a putative target sequence.
利用激动性抗CD40单克隆抗体将肽靶向抗原呈递细胞已成功用作增强免疫反应的策略。在鸡身上进行测试时，抗体引导平台能够在免疫后1周内诱导特异性IgG产生。然而，这种方法超出其最初作为疫苗递送工具的概念的应用尚未得到充分开发。在本研究中，α毒素被用作模型微生物毒素进行表位作图，通过抗体引导免疫方法产生一组针对毒素特定区域的抗体，试图确定毒素上的关键决定簇，一旦结合，将阻碍下游毒性。α毒素具有溶血和磷脂酶C（PLC）酶活性，长期以来一直被认为是家禽坏死性肠炎疾病的关键破坏性病原体之一。先前使用昂贵且劳动密集的定点诱变技术来鉴定介导其酶活性的毒素上的关键抗原决定簇。为了创建一组抗体，使用公共领域的B细胞表位预测算法选择了23个短的候选α毒素肽区域，并进行定制合成，以加载到抗体引导复合物上用于在鸟类中免疫以产生抗血清。针对所有候选中和表位产生了肽特异性抗体反应，并用于毒素中和试验。针对所有23种肽的抗血清都能够中和毒素的溶血活性，中和效价范围为80至320，但没有一种能有效阻断PLC。抗体引导免疫的新方法引入了一种新的、廉价的方法来生产多克隆IgG，并在从推定的靶序列分析开始的2周内鉴定微生物毒素和酶中的中和表位。
