Section of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United Kingdom.
Flow Cytometry Core Facility, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Front Immunol. 2023 Jun 2;14:1184510. doi: 10.3389/fimmu.2023.1184510. eCollection 2023.
Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, and . Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned mICA versus ~53% cloned the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery.
表达克隆全人源单克隆抗体（hmAb）在疫苗学领域具有强大的应用价值，特别是在阐明疫苗诱导的 B 细胞反应和新型疫苗候选抗原发现方面。hmAb 克隆过程的准确性依赖于高效分离感兴趣的 hmAb 产生浆母细胞。先前，开发了一种新型免疫球蛋白捕获测定（ICA），使用单一蛋白疫苗抗原，以提高病原体特异性 hmAb 克隆产量。在这里，我们报告了该单抗原 ICA 的一种新型修饰，使用福尔马林处理的、荧光染色的人类细菌侵袭性病原体的全细胞混悬液，和 。通过形成抗 CD45-链霉亲和素和生物素抗 IgG 支架，实现了个体疫苗抗原特异性浆母细胞分泌的 IgG 的隔离。然后，在单细胞分选过程中，使用含有异源肺炎球菌和脑膜炎球菌菌株的混悬液分别富集多糖和蛋白抗原特异性浆母细胞。应用改良的全细胞 ICA（mICA）后，与使用标准（非 mICA）方法获得的 19/31（61%）抗肺炎球菌多糖 hmAb 相比，克隆了 14/59（14%）抗肺炎球菌多糖 hmAb - 代表 hmAb 克隆精度提高了约 4.4 倍。对于抗脑膜炎球菌疫苗 hmAb 克隆，获得了更为适度的1.7 倍差异；mICA 克隆的88% hmAb 针对脑膜炎球菌表面蛋白，而标准方法克隆的~53% hmAb 针对脑膜炎球菌表面蛋白。VDJ 测序显示，克隆的 hmAb 反映了对肺炎球菌和脑膜炎球菌疫苗的回忆反应；hmAb 克隆内的多样化是通过对替换突变的阳性选择发生的。因此，我们已经证明了在 ICA 方案中成功利用全细菌细胞能够分离针对多个不同表位的 hmAb，从而增强了反向疫苗学 2.0（RV 2.0）等方法的效力，用于细菌疫苗抗原发现。
