Clonal analysis of a human antibody response: II. Sequences of the V(H) genes of human IgM, IgG, and IgA to rabies virus reveal preferential utilization of V(H)III 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 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 V(H) gene segments of the V(H)III family. The remaining two IgG1 mAb utilized gene segments of the V(H)I and V(H)IV families. Of the seven expressed V(H)III family genes, three were similar to the germline V(H)26c gene, two to the germline 22-2B gene, one to the germline H11 gene, and one to the germline 8- 1B gene. The expressed V(H)I and V(H)IV 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) 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 J(H) segment utilization was unbalanced and reminiscent of those of the adult and fetus. Four mAb utilized J(H)4, two J(H)6, two J(H)3, and one J(H)5; no mAb utilized J(H)1 or J(H)2 genes. The present data suggest that the adult human Ig 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 Ig V gene hypermutation in human B cells producing high affinity antibodies.