Isolation of human monoclonal antibodies from 4CMenB vaccinees reveals PorB and LOS as the main OMV components inducing cross-strain protection [ Recombinant Chip]

In this study, we describe the development and use of an ad hoc protein microarray to characterize the target of HumAbs isolated from 4CMenB vaccinated subjects and induced by the OMV component of the vaccine A panel of recombinant meningococcal proteins were printed randomly in replicates on a not-commercial protein microarray. With the expection of NadA, GNA2091-fHbp and NHBA-GNA1030 (that are tagless), all the proteins were expressed as FLAG- and His-tagged fusions. Recombinant antigens were expressed in E. coli and then purified either from the cytoplasmic fraction as soluble proteins or resolubilized from the inclusions bodies as insoluble forms. Controls consisted of 8 serial 2-fold dilutions of human IgG, proteins expressed and purified from E. coli following the same expression and purification protocol, but originating from different pathogens other than MenB and PBS + 40% glycerol spots.

本研究详述了一款定制化蛋白质微阵列的开发与应用，该微阵列用于表征从接种4CMenB疫苗的受试者体内分离得到的、由该疫苗外膜囊泡（Outer Membrane Vesicle，OMV）组分诱导产生的人源抗体（Human Antibodies，HumAbs）的靶向抗原。一组重组脑膜炎球菌蛋白以重复随机点样的方式制备于非商用蛋白质微阵列上。除NadA、GNA2091-fHbp与NHBA-GNA1030这三种无标签蛋白外，其余蛋白均以FLAG标签与His标签融合蛋白的形式进行表达。重组抗原在大肠杆菌（Escherichia coli，E. coli）中表达，随后或从细胞质组分中以可溶性蛋白形式纯化，或从包涵体中以不溶性形式复溶纯化。对照组包含8个连续两倍稀释梯度的人免疫球蛋白G（Immunoglobulin G，IgG）、按照相同表达纯化流程从大肠杆菌中表达纯化得到的非脑膜炎奈瑟菌（MenB）来源的其他病原体蛋白，以及添加40%甘油的磷酸盐缓冲液点样对照。