Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120

Abstract

IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.

Involvement of FcγR I (CD64) in the Mechanism of HIV-1 Inhibition by Polyclonal IgG Purified from Infected Patients in Cultured Monocyte-Derived Macrophages

“…These mAbs have been described to exhibit broad cross-neutralizing activities against a wide range of primary isolates of HIV-1 in vitro in assays involving PBMC as target cells (27)(28)(29)(30)(31)(32). Stamatatos et al (33) have already described a similar 10-fold increase in the inhibition of MDM infection for mAb IgG1b12.…”

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

“…An examination of antibody binding site structures has revealed three main topographies, cavity, grooved, and flat, binding haptens, peptides, and proteins, respectively (77)(78)(79)(80)(81)(82), with rare antibodies showing alternative topographies such as long finger-like HCDR3 projections (83,84). With the exception of polypeptide modifications, such as ubiquitination (85), most PTMs can be considered to be small haptens.…”

The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

“…They include the gp41 Abs 2F5 and 4E10 and the gp120 Abs 2G12 and b12 (12)(13)(14)(15)(16)(17). Data derived from the crystal structure of envelope-Ab complexes suggest that the neutralizing activity of these Abs (except for 2G12) is mediated by an unusually long CDR H3 loop that penetrates deeply into the Ag cleft, which is obscured in the heterotrimeric envelope (12,15,18). Unfortunately, broad HIV neutralizing Abs occur only rarely in patients, while experimental vaccines have failed to induce a significant Ab response to autologous primary isolates (19 -25) let alone nonautologous strains (26,27).…”