Clonal analysis of a human antibody response - II. Sequences of the V_H genes of human IgM, IgGIgA to rabies virus reveal preferential utilization of V_HIII segments and somatic hypermutation

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 V_H regions. Six mAb (five lgG1 and one lgA1) were monoreactive and displayed high affinities for rabies virus Ag. The remaining three mAb (lgM) were polyreactive and displayed lower affinities for rabies virus Ag. Seven mAb (3 lgG1, the lgA1the three lgM) utilized V_H gene segments of the V_HIII family. The remaining two lgG1 mAb utilized gene segments of the V_HI and V_HIV families. Of the seven expressed V_HIII family genes, three were similar to the germline V_H26c gene, two to the germline 22-2B gene, one to the germline H11 geneone to the germline 8-1B gene. The expressed V_HI and V_HIV genes displayed sequences similar to those of the germline hv1263 and V71-4 genes, respectively. The V_H genes of all but one mAb (mAb55) resembled those that are predominantly expressed by Cμ⁺ clones in human fetal liver libraries. When compared with known germline sequences, the V_H 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 V_H gene of the high affinity (anti-rabies virus glycoprotein) lgG1 mAb57 that has been shown to efficiently neutralize the virus in vitro and in vivo. The distribution, mainly within the complementarity determining regionsthe 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 J_H segment utilization was unbalanced and reminiscent of those of the adult and fetus. Four mAb utilized J_H4, two J_H6, two J_H3one J_H5; no mAb utilized J_H1 or J_H2 genes. The present data suggest that the adult human lg V gene assortment expressed as the result of selection by a proteinic mosaic Ag is more restricted than previously assumed and resembles that of the putatively unselected adult B cell repertoire and the unselected Cμ⁺ cell repertoire of the fetus. They also document somatic lg V gene hypermutation in human B cells producing high affinity antibodies.