Lei Lin, Tran Karen, Wang Yimeng, Steinhardt James J, Xiao Yongli, Chiang Chi-I, Wyatt Richard T, Li Yuxing
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.
IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, United States.
Front Microbiol. 2019 Apr 23;10:672. doi: 10.3389/fmicb.2019.00672. eCollection 2019.
Here, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for analyzing immunization- or viral infection-elicited antibody response at the clonal level in guinea pigs. We stained the peripheral blood mononuclear cells (PBMCs) from a guinea pig immunized with HIV-1 envelope glycoprotein trimer mimic (BG505 SOSIP), using anti-guinea pig IgG and IgM fluorochrome conjugates, along with fluorochrome-conjugated BG505 SOSIP trimer as antigen (Ag) probe to sort for Ag-specific IgG IgM B cells at single cell density. We then designed a set of guinea pig immunoglobulin (Ig) gene-specific primers to amplify cDNAs encoding B cell receptor variable regions [V(D)J segments] from the sorted Ag-specific B cells. B cell V(D)J sequences were verified by sequencing and annotated by IgBLAST, followed by cloning into Ig heavy- and light-chain expression vectors containing human IgG1 constant regions and co-transfection into 293F cells to reconstitute full-length antibodies in a guinea pig-human chimeric IgG1 format. Of 88 antigen-specific B cells isolated, we recovered 24 (27%) cells with native-paired heavy and light chains. Furthermore, 85% of the expressed recombinant mAbs bind positively to the antigen probe by enzyme-linked immunosorbent and/or BioLayer Interferometry assays, while five mAbs from four clonal lineages neutralize the HIV-1 tier 1 virus ZM109. In summary, by coupling Ag-specific single B cell sorting with gene-specific single cell RT-PCR, our method exhibits high efficiency and accuracy, which will facilitate future efforts in isolating mAbs and analyzing B cell responses to infections or immunizations in the guinea pig model.
在此，我们建立了一个抗原特异性单B细胞分选和单克隆抗体（mAb）克隆平台，用于在豚鼠中克隆水平分析免疫或病毒感染引发的抗体反应。我们用抗豚鼠IgG和IgM荧光染料偶联物，以及荧光染料偶联的BG505 SOSIP三聚体作为抗原（Ag）探针，对用HIV-1包膜糖蛋白三聚体模拟物（BG505 SOSIP）免疫的豚鼠外周血单个核细胞（PBMC）进行染色，以单细胞密度分选Ag特异性IgG IgM B细胞。然后，我们设计了一组豚鼠免疫球蛋白（Ig）基因特异性引物，从分选的Ag特异性B细胞中扩增编码B细胞受体可变区[V（D）J片段]的cDNA。通过测序验证B细胞V（D）J序列，并通过IgBLAST进行注释，随后克隆到包含人IgG1恒定区的Ig重链和轻链表达载体中，并共转染到293F细胞中，以豚鼠-人嵌合IgG1形式重建全长抗体。在分离的88个抗原特异性B细胞中，我们回收了24个（27%）具有天然配对重链和轻链的细胞。此外，85%的表达重组mAb通过酶联免疫吸附和/或生物层干涉测定法与抗原探针呈阳性结合，而来自四个克隆谱系的五个mAb可中和HIV-1 1级病毒ZM109。总之，通过将Ag特异性单B细胞分选与基因特异性单细胞RT-PCR相结合，我们的方法具有高效率和准确性，这将有助于未来在豚鼠模型中分离mAb以及分析B细胞对感染或免疫的反应。
