Alam S

Alam S. genetic and structural factors that could affect antigen affinity of unmutated na?ve B cell receptors. Here, we studied allelic variants of the VH2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both VH2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the NH54 variant for fusion-intermediate conformation was an order of magnitude lower than that of the DH54 VH2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design. INTRODUCTION The human immunoglobulin (Ig) VH (heavy-chain variable region) genes are grouped into 7 families (VH1 to VH7) (4, 24, 30) that are not equally represented in the human B cell repertoire (7, 8, 39). While VH gene usage in cord blood lymphocytes reflects the relative frequency of VH family size, some VH subfamily genes are expressed at higher frequency (14, 20, 38). The control of VH expression by genetic factors has been described in studies on monozygotic twins (19, 41), and VH polymorphisms have been associated with autoimmune diseases (40, 41, 45, 47). VH gene expression, shaped by both genetic and antigenic stimulation, can also regulate the ability of the host to induce an antibody response against a pathogen. The HIV-1 envelope protein has conserved regions in gp41 to which rare broadly neutralizing human antibodies like 2F5 and 4E10 have been isolated (5, 27), However, only 10 to 20% of chronically infected subjects make broadly neutralizing antibodies (bNAbs) (32, 43). Even when bNAbs are made, they are made not during the acute infection but rather only after months of chronic infection (15, 32, 34). Viral factors contributing to difficulty in inducing bNAbs include conformational masking of transient epitopes (3, 21) as well as glycan shielding (31, 44). HIV-1 gp41 bNAbs are more uncommon than those that target gp120, with one hypothesis being that lack of induction of gp41 Procyanidin B2 neutralizing antibodies is due to viral mimicry of the gp41 membrane proximal external region (MPER), with host molecules leading to tolerance to gp41 bNAb induction (3, 16, 17, 42). However, the nature of host factors regulating the ability to make HIV-1 Env bNAbs remains unknown. Xiao et al. have suggested the lack of antigen recognition by unmutated receptors on na?ve B cells, therefore implying that there may be holes in the human germ line B cell repertoire for antigens that stimulate na?ve B cells that can make broadly neutralizing MTC1 antibodies, and in particular those that can make 2F5-like gp41 bNAbs (46). The 2F5 bNAb (25, 29) uses the VH2-5 gene, for which 10 distinct alleles are known. Each uses either a D or an N residue at position H54 (22), and the relative frequencies of human VH2-5-bearing antibodies that use N or D alleles are 54% and 46%, respectively (Table 1). The mature 2F5 bNAb usage of VH2-5 includes the DH54 residue, which resides in the heavy-chain complementarity-determining region 2 (HCDR2) (27) (Fig. 1). Since the 2F5 HCDR2 DH54 residue makes an important contact (salt bridge with Procyanidin B2 the 2F5 core epitope residue 665K) with the antigen, structural considerations suggested that the variant with HCDR2 NH54 is likely to have a weaker affinity for antigen. Thus, genetic and structural factors could affect antigen affinity of unmutated na?ve B cell receptors (BCR) and induction of 2F5-like antibodies. In this study, we describe the antigen reactivity of the two allelic variants of the VH2-5 inferred germ line variant of the 2F5 bNAb and show that, while both the variant putative germ line antibodies bind to gp41 antigens, their affinities to the gp41-inter protein (13), a mimic of the putative fusion intermediate conformation, differed Procyanidin B2 by an order of magnitude and were determined by the HCDR2 residues NH54/DH54, which define allelic variants. Open in a separate window Fig. 1. Bonding between 2F5 HCDR2 DH54 and gp41 MPER 665K (27). The contact surface of 2F5 bound to its antigenic peptide shows a strong complementarity Procyanidin B2 of charge, and two CDR H2 residues, DH56 and DH54, interact through hydrogen bonds and salt bridges with K665 of the 2F5 core tripeptide DKW. Based on the CDR H2 substitutions in the two UAs, the.