<i>In silico</i> site-directed mutagenesis of neutralizing mAb 4C4 and analysis of its interaction with G-H loop of VP1 to explore its therapeutic applications against FMD

Investigating the behaviour of bio-molecules through computational mutagenesis is gaining interest to facilitate the development of new therapeutic solutions for infectious diseases. The antigenetically variant genotypes of foot and mouth disease virus (FMDV) and their subsequent infections are challenging to tackle with traditional vaccination. In such scenario, neutralizing antibodies might provide an alternate solution to manage the FMDV infection. Thus, we have analysed the interaction of the mAb 4C4 with a synthetic G-H loop of FMDV-VP1 through <i>in silico</i> mutagenesis and molecular modelling. Initially, a set of 25,434 mutants were designed and the mutants having better energetic stability than 4C4 were clustered based on sequence identity. The best mutant representing each cluster was selected and evaluated for its binding affinity with the antigen in terms of docking scores, interaction energy and binding energy. Six mutants have confirmed better binding affinities towards the antigen than 4C4. Further, interaction of these mutants with the natural G-H loop that is bound to mAb SD6 was also evaluated. One 4C4 variant having mutations at the positions 2034(N→L), 2096(N→C), 2098(D→Y), 2532(T→K) and 2599(A→G) has revealed better binding affinities towards both the synthetic and natural G-H loops than 4C4 and SD6, respectively. A molecular dynamic simulation for 50 ns was conducted for mutant and wild-type antibody structures which supported the pre-simulation results. Therefore, these mutations on mAb 4C4 are believed to provide a better antibody-based therapeutic option for FMD. Communicated by Ramaswamy H. Sarma借助计算诱变（computational mutagenesis）技术探究生物分子的行为特征，正成为推动传染病新型治疗方案研发的研究热点。口蹄疫病毒（foot and mouth disease virus, FMDV）的抗原变异基因型及其引发的后续感染，难以通过传统疫苗接种策略实现有效防控。在此背景下，中和抗体或可为口蹄疫病毒感染的防控提供替代解决方案。 本研究采用虚拟（in silico）诱变与分子建模技术，分析了单克隆抗体4C4（monoclonal antibody 4C4, mAb 4C4）与FMDV-VP1蛋白合成G-H环的相互作用：研究初期共设计25434个突变体，基于序列同一性将能量稳定性优于原始4C4的突变体进行聚类；选取每个聚类中的最优突变体，从对接得分、相互作用能及结合能三个维度，评估其与抗原的结合亲和力。经验证，共有6个突变体对该抗原的结合亲和力优于原始4C4抗体。 进一步评估了上述突变体与结合了单克隆抗体SD6（mAb SD6）的天然G-H环的相互作用。结果显示，在2034位（天冬酰胺→亮氨酸，N→L）、2096位（天冬酰胺→半胱氨酸，N→C）、2098位（天冬氨酸→酪氨酸，D→Y）、2532位（苏氨酸→赖氨酸，T→K）及2599位（丙氨酸→甘氨酸，A→G）位点引入突变的4C4变体，对合成型与天然型G-H环的结合亲和力分别优于原始4C4与SD6抗体。 随后对该突变体及野生型（wild-type）抗体结构开展了50纳秒的分子动力学模拟，模拟结果验证了预实验的分析结论。因此，对单克隆抗体4C4引入上述突变，有望为口蹄疫提供更优的抗体类治疗方案。本文由Ramaswamy H. Sarma转交刊发。