Clonal analysis of a human antibody response. II. Sequences of the VH genes of human IgM, IgG, and IgA to rabies virus reveal preferential utilization of VHIII segments and somatic hypermutation

Abstract

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)

FIGURE 1

Nucleotide (A) and deduced amino acid (B) sequences of the V_H genes utilized by the mAb binding to rabies virus. In each cluster, the top sequence is given for comparison and represents the published germline V_H gene displaying the highest degree of identity to the expressed V_H genes. The VH26c, 22-2B, H11, and 8-1B V_H genes belong to the V_HIII family. V71-4 and hv1263 are members of the V_HIV and V_HI gene families, respectively. Dashes indicate identities. Solid lines on the top of each cluster depict CDR. Small letters denote 5′ untranslated sequences (UT) and, in the case of hv1263 and 57GTA8, introns. 52G25 is an unpublished germline sequence (see text). 57GTA8 is the germline sequence we amplified from PMN DNA of the subject whose B cells were used for the generation of mAb57. The sequences or complementary sequences of the primers adopted for genomic V_H gene amplification are underlined. 30p1, 60p2, 58p2, and 51p1 are V_H genes expressed in fetal liver (35). Their nucleotide and deduced amino acid sequences are used for comparison. The present sequences are available from EMBL/GenBank/DDBJ under accession numbers L-08082, L-08083, L-08084, L-08085, L-08086, L-08087, L-08088, L-08089 and L-08090.

FIGURE 1

Nucleotide (A) and deduced amino acid (B) sequences of the V_H genes utilized by the mAb binding to rabies virus. In each cluster, the top sequence is given for comparison and represents the published germline V_H gene displaying the highest degree of identity to the expressed V_H genes. The VH26c, 22-2B, H11, and 8-1B V_H genes belong to the V_HIII family. V71-4 and hv1263 are members of the V_HIV and V_HI gene families, respectively. Dashes indicate identities. Solid lines on the top of each cluster depict CDR. Small letters denote 5′ untranslated sequences (UT) and, in the case of hv1263 and 57GTA8, introns. 52G25 is an unpublished germline sequence (see text). 57GTA8 is the germline sequence we amplified from PMN DNA of the subject whose B cells were used for the generation of mAb57. The sequences or complementary sequences of the primers adopted for genomic V_H gene amplification are underlined. 30p1, 60p2, 58p2, and 51p1 are V_H genes expressed in fetal liver (35). Their nucleotide and deduced amino acid sequences are used for comparison. The present sequences are available from EMBL/GenBank/DDBJ under accession numbers L-08082, L-08083, L-08084, L-08085, L-08086, L-08087, L-08088, L-08089 and L-08090.

FIGURE 2

Nucleotide (A) and deduced amino acid (B) sequences of the D and J_H segments of the mAb binding to rabies virus. Germline D genes are given for comparison. Dashes indicate identities. Inverted DXP4 sequence is the reverse strand of the germline DXP4 sequence. The present sequences are available from EMBL/GenBank/DDBJ under accession numbers L-08082 through L-08090.

FIGURE 3

PCR analysis of somatic mutations in the expressed mAb57 V_H gene. A, Ethidium bromide staining of amplified DNA fractionated in agarose gel electrophoresis (10 μl of reaction mixture were applied to each lane). Using the CDR1 (57CR1) and FR3 sequence (HI-7) oligonucleotide primers (see Materials and Methods), an amplification product of appropriate size (last 3′ portion of the V_H segment, about 200 bp) was obtained by priming DNA from mAb57 B cells (hybridoma DNA) but not the DNA from autologous PMN (PMN DNA). B, Ethidium bromide staining of amplified DNA-fractionated in agarose gel electrophoresis (10 μl of reaction mixture were applied to each lane). Using the leader (HI-6) and FR3 (HI-7) sequence oligonucleotide primers (see Materials and Methods), amplification products of identical and appropriate size (about 400 bp) were obtained by priming DNA from both mAb57 B cells (hybridoma DNA) and autologous PMN (PMN DNA). C, Southern blot hybridization of the PCR products shown in B with the ³²P-labeled oligonucleotide probe encompassing the CDR1 sequence of the expressed mAb57 V_H gene (see Materials and Methods).