Ikematsu H, Harindranath N, Ueki Y, Notkins A L, Casali P
Department of Pathology, New York University School of Medicine, New York 10016.
J Immunol. 1993 Feb 15;150(4):1325-37.
The construction of mAb-producing cell lines has been instrumental in dissecting the fine specificities and genetic makeup of murine antibodies to exogenous and self Ag. The analysis of the genetic composition of human antibody responses has been hampered by the difficulty in generating human mAb of predetermined class and specificity. Using B lymphocytes from three healthy subjects vaccinated with inactivated rabies virus vaccine, we generated nine human mAb binding to rabies virus and analyzed the genes encoding their VH regions. Six mAb (five IgG1 and one IgA1) were monoreactive and displayed high affinities for rabies virus Ag. The remaining three mAb (IgM) were polyreactive and displayed lower affinities for rabies virus Ag. Seven mAb (3 IgG1, the IgA1, and the three IgM) utilized VH gene segments of the VHIII family. The remaining two IgG1 mAb utilized gene segments of the VHI and VHIV families. Of the seven expressed VHIII family genes, three were similar to the germline VH26c gene, two to the germline 22-2B gene, one to the germline H11 gene, and one to the germline 8-1B gene. The expressed VHI and VHIV genes displayed sequences similar to those of the germline hv1263 and V71-4 genes, respectively. The VH genes of all but one mAb (mAb55) resembled those that are predominantly expressed by C mu + clones in human fetal liver libraries. When compared with known germline sequences, the VH genes of the rabies virus-binding mAb displayed variable numbers of nucleotide differences. That such differences resulted from a process of somatic hypermutation was formally demonstrated (by analyzing DNA from polymorphonuclear neutrophil of the same subject whose B lymphocytes were used for the mAb generation) in the case of the VH gene of the high affinity (anti-rabies virus glycoprotein) IgG1 mAb57 that has been shown to efficiently neutralize the virus in vitro and in vivo. The distribution, mainly within the complementarity determining regions, and the high replacement-to-silent ratio of the mutations, were consistent with the hypothesis that the mAb57-producing cell clone underwent a process of Ag-driven affinity maturation through clonal selection. The D gene segments of the rabies virus-selected mAb were heterogeneous and, in most cases, flanked by significant N segment additions. The JH segment utilization was unbalanced and reminiscent of those of the adult and fetus. Four mAb utilized JH4, two JH6, two JH3, and one JH5; no mAb utilized JH1 or JH2 genes.(ABSTRACT TRUNCATED AT 400 WORDS)
单克隆抗体产生细胞系的构建对于剖析鼠源抗体对外源和自身抗原的精细特异性及基因组成起到了重要作用。人类抗体应答的基因组成分析因难以产生具有预定类别和特异性的人源单克隆抗体而受到阻碍。我们使用来自三名接种灭活狂犬病病毒疫苗的健康受试者的B淋巴细胞，产生了九种与人狂犬病病毒结合的人源单克隆抗体，并分析了编码其重链可变区（VH）的基因。六种单克隆抗体（五种IgG1和一种IgA1）具有单一反应性，对狂犬病病毒抗原有高亲和力。其余三种单克隆抗体（IgM）具有多反应性，对狂犬病病毒抗原的亲和力较低。七种单克隆抗体（3种IgG1、IgA1和3种IgM）利用了VHIII家族的VH基因片段。其余两种IgG1单克隆抗体利用了VHI和VHIV家族的基因片段。在七个表达的VHIII家族基因中，三个与胚系VH26c基因相似，两个与胚系22 - 2B基因相似，一个与胚系H11基因相似，一个与胚系8 - 1B基因相似。表达的VHI和VHIV基因分别显示出与胚系hv1263和V71 - 4基因相似的序列。除一种单克隆抗体（mAb55）外，所有单克隆抗体的VH基因都类似于人胎儿肝脏文库中Cμ +克隆主要表达的基因。与已知胚系序列相比，狂犬病病毒结合单克隆抗体的VH基因显示出不同数量的核苷酸差异。在高亲和力（抗狂犬病病毒糖蛋白）IgG1单克隆抗体mAb57的VH基因情况下（通过分析来自与用于产生单克隆抗体的B淋巴细胞相同受试者的多形核中性粒细胞的DNA），正式证明了这种差异是由体细胞超突变过程导致的，该单克隆抗体已被证明在体外和体内均能有效中和病毒。突变主要分布在互补决定区，且替换与沉默的比例较高，这与产生mAb57的细胞克隆通过克隆选择经历抗原驱动的亲和力成熟过程的假设一致。狂犬病病毒选择的单克隆抗体的D基因片段是异质的，并且在大多数情况下两侧有明显的N片段添加。JH片段的利用不均衡，类似于成人和胎儿的情况。四种单克隆抗体利用JH4，两种利用JH6，两种利用JH3，一种利用JH5；没有单克隆抗体利用JH1或JH2基因。（摘要截短至400字）
