Structure of the VH and VL segments of polyreactive and monoreactive human natural antibodies to HIV-1 and Escherichia coli beta-galactosidase

Abstract

B lymphocytes committed to the production of antibodies binding to antigens on pathogenic bacteria and viruses (natural antibodies) are common components of the normal human B cell repertoire. A major proportion of natural antibodies is capable of binding multiple antigens (polyreactive antibodies). Using B cells from three HIV-1 seronegative healthy subjects, and purified HIV-1 and beta-galactosidase from Escherichia coli as selecting antigen, we generated three natural IgM mAb to HIV-1 and a natural IgM mAb to beta-galactosidase. The three HIV-1-selected antibodies (mAb102, mAb103, and mAb104) were polyreactive. They bound with different affinities (Kd = 10(-6) to 10(-8) M) to the HIV-1 envelope gp160, the p24 core protein, and the p66 reverse transcriptase, but not to the 120 glycosylated env protein. They also bound to beta-galactosidase (Kd approximately 10(-7) M), tetanus toxoid, and various various self antigens. In contrast, the natural mAb selected for binding to beta-galactosidase (mAb207.F1) was monoreactive, in that it bound with a high affinity (Kd < 10(-8) M) to this antigen, but to none of the other antigens tested, including HIV-1. Structural analysis of the VH and VL segments revealed that the natural mAb utilized three segments of the VHIV gene family and one of the VHIII family, in conjunction with VL segments of the V lambda I, V lambda II, V lambda III, or V kappa IV subgroups. In addition, the natural mAb VH and VL segments were in unmutated or virtually unmutated (germline) configuration, including those of the monoreactive mAb207.F1 to beta-galactosidase, and were identical or closely related to those utilized by specific autoantibodies or specific antibodies to viral and/or bacterial pathogens. Thus, the present data show that both polyreactive and monoreactive natural antibodies to foreign antigen can be isolated from the normal human B cell repertoire. They also suggest that the VH and VL segments of not only polyreactive but also monoreactive natural antibodies can be encoded in unmutated or minimally mutated genes, and possibly provide the templates for the specific high affinity antibodies elicited by self or foreign antigens.

Fig. 1

Binding of natural human mAb to HIV-1 (◇), tetanus toxod (■), β-galactosidase from E. coli (+), ssDNA (△), insulin (●), thyroglobulin (▲), Fc fragment of human IgG (○), and BSA (□).

Fig. 2

Nucteotide (A) and deduced amino acid (B) sequences of the V_H genes utilized by the natural human mAb. In each duster, the top sequence is given for comparison and represents the germline V_H gene displaying the highest degree of identity to the expressed V_H genes of the cluster The 4.33, V_H4.2 1 , and 3d279d genes belong to the V_HIV family. The leader region sequence of the V_H4.21 is not available, that of the V58 gene leader region is provided instead. The V_H26c gene is a member of the V_HIII family. Dashes indicate identities. Solid lines on the top of each duster depict CDR. Small letters denote untranslated sequences Asterisks indicate translation initiation codons. The present sequences are available form EMBL/GenBank/DDBJ under accession numbers L25291, L25292, L25293, and L25294.

Fig. 3

Nudeotide (A) and deduced amino acid (B) sequences of the natural mAb D and J_H segments. Germline D genes are given for comparison. Dashes indicate identities. The present sequences are available from EMBUGenBank/DDBJ under accession numbers L25291, L25292, L25293, and L25294.

Fig. 4

Nucleotide (A) and deduced amino add (B) sequences of the V_L genes utilized by the natural human mAb. In each duster, the top sequence is given for comparison and represents the published V_L gene displaying the highest degree of homotogy to the expressed V_L genes of the cluster. V_λIII.1 (V_λIII subgroup), V_λIV, and Hum1v117 (V_λI subgroup) are sequences of genomic germline genes. T2:C5 (V_λI subgroup) and DSC V_λII are sequences rearranged, but possibly unmutated genes. Dashes ridicate identities. Solid lines on the top of each duster depict complimentarily determining regions. Small letters denote untranslated sequences. Asterisks indicate translation initiation codons. The present sequences are available from EMBL/GenBank/DDBJ under accession numbers L25295, L25296, L25297, and L25298.

Fig. 5

Nucleobde (A) and deduced amino acid (B) sequences of the J_L segments of the natural mAb. In each cluster, the top sequence is given for comparison and represents the published germline J_L gene displaying the highest degree of homology to the expressed J_L genes of the cluster. Dashes indicate identities. The present sequences are available from EMBUGenBank/DDBJ under accession numbers L25295, L25296, L25297, and L25298.