Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

中文翻译：

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体外预筛选尼古丁半抗原连接体，以选择用于体内药代动力学评价的半抗原结合疫苗候选物。


自从尼古丁疫苗被证明可以作为治疗烟草烟雾影响的一种可能的治疗干预措施以来，人们为增强尼古丁特异性免疫力做出了广泛的努力。尼古丁半抗原的接头修饰一直是提高尼古丁免疫原性的焦点，其中这些修饰的评估通常依赖于体内动物模型，例如小鼠、大鼠或非人灵长类动物。在这里，我们提出了两种体外筛选策略来估计和预测我们新设计的尼古丁半抗原的免疫原性潜力。一种方法是利用竞争酶联免疫吸附测定 (ELISA) 来分析尼古丁半抗原或半抗原蛋白缀合物与尼古丁特异性抗体（多克隆和单克隆）的相互作用。另一种方法依赖于半抗原和载体蛋白缀合位点附近的氨基酸残基之间相互作用的计算模型，以推断接头-载体蛋白缀合对抗尼古丁抗体反应的影响。使用这两种体外方法，我们对具有不同接头的半抗原作为可行候选疫苗的潜力进行了排名。基于 ELISA 的半抗原排名与体内尼古丁药代动力学分析获得的结果一致。研究发现，半抗原与抗 Nic 单克隆抗体的平均结合亲和力 (IC50) 与免疫小鼠的平均脑尼古丁浓度之间存在相关性。半抗原和载体蛋白相互作用的计算模型有助于排除具有强接头-载体缀合效应和低体内功效的缀合物。这些体外筛选策略的简单性应有助于选择和开发更有效的尼古丁结合疫苗。 此外，这些数据强调了接头和半抗原蛋白缀合物由于包含在结合和激活 B 细胞的表位中而对缀合疫苗免疫原性的贡献，这一贡献先前未被充分认识。