Structural Basis for HIV-1 Neutralization by 2F5-Like Antibodies m66 and m66.6

Antibodies m66.6 and 2F5 are the only effective human HIV-1-neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein. Although 2F5 has been extensively characterized, much less is known about antibody m66.6 or antibody m66, a closely related light-chain variant. Here, we report the crystal structure of m66 in complex with its gp41 epitope, along with unbound structures of m66 and m66.6. We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp41 and determined the molecular basis that underlies their neutralization of HIV-1. When bound by m66, the N-terminal region of the gp41 MPER adopts a conformation comprising a helix, followed by an extended loop. Comparison of gp41-bound m66 to unbound m66.6 identified three light-chain residues of m66.6 that were confirmed through mutagenesis to underlie the greater breadth of m66.6-mediated virus neutralization. Recognition of gp41 by m66 also revealed similarities to antibody 2F5 both in the conformation of crucial epitope residues as well as in the angle of antibody approach. Aromatic residues at the tip of the m66.6 heavy-chain third complementarity-determining region, as in the case of 2F5, were determined to be critical for virus neutralization in a manner that correlated with antibody recognition of the MPER in a lipid context. Antibodies m66, m66.6, and 2F5 thus utilize similar mechanistic elements to recognize a common gp41-MPER epitope and to neutralize HIV-1.     

Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region

The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.The membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane glycoprotein is the target of three broadly neutralizing anti-HIV-1 antibodies, 2F5, Z13e, and 4E10, and is thus a potential site of HIV-1 vulnerability to the humoral immune response (21, 24, 27, 48). The MPER encompasses ∼25 residues at the carboxyl-terminal end of the predicted gp41 ectodomain, just before the transmembrane region, and is rich in aromatic residues, typical of bilayer-interfacial regions of membrane proteins (26, 36, 40). Mutation of selected MPER tryptophans abrogates gp41-mediated fusion of the viral and target cell membranes, indicating that this region is crucial for HIV-1 infectivity (23, 28). Structural studies of unbound forms of the gp41 MPER both in solution and in lipid contexts have demonstrated that it adopts a number of conformations, many of which are α-helical, and electron-paramagnetic resonance measurements have indicated lipid bilayer immersion depths for MPER residues that range from acyl to phospholipid headgroup regions (4, 7, 8, 19, 32, 37). The binding of neutralizing antibodies, such as 2F5, to the MPER must therefore account for the membrane milieu in which the epitope is found.The 2F5 antibody has been shown to exhibit ∼100-fold-enhanced binding to its epitope on uncleaved gp140s when presented in the context of lipid proteoliposomes (11, 25), and other studies have shown that 2F5 can contact phospholipids directly in the absence of gp41 (1, 3, 12, 22, 29, 30). The latter finding has led to the suggestion that 2F5 might be autoreactive (12), although passive transfusion of 2F5 does not appear to have deleterious effects (38) and 2F5 failed to react in some clinically based assays for autoreactive lipid antibodies (31, 39). The crystal structures of the 2F5 antibody in complex with its gp41 MPER epitope revealed that, despite the 22-residue length of the 2F5 heavy chain third complementarity-determining region (CDR H3) loop, contacts with the gp41 MPER peptide are made predominantly at the loop base. In some crystal structures, the tip of the loop protrudes away from gp41, while in others, it is disordered (9, 14, 25). A unique feature of the tip of the CDR H3 loop is that it contains a patch of hydrophobic residues, including residues L100_A, F100_B, V100_D, and I100_F (Kabat numbering), which, with the exception of I100_F, do not contact gp41 (9, 10, 14, 25) (Fig. ​(Fig.1).1). While a prior study revealed the importance of residue F100_B of the CDR H3 loop in 2F5-neutralizing activity, nonconservative residue substitutions at this position also appeared to diminish 2F5 binding to the immobilized MPER peptide and gp41 in enzyme-linked immunosorbent assay (ELISA) formats (47). Conversely, a more recent study has shown that alanine mutations in the 2F5 CDR H3 loop can affect neutralization without affecting gp41 binding (2).Open in a separate windowFIG. 1.2F5 CDR H3 loop mutagenesis. (A) Structure of 2F5 Fab (blue and gray) in complex with a gp41 peptide (red). The 2F5 CDR H3 (purple) contacts gp41 only at its base, while the tip extends away from the peptide. (B) Close-up view of the 2F5 CDR H3 loop, with hydrophobic residues at the loop tip shown in stick representation and colored green. (C) Mutations introduced into the tip of the 2F5 CDR H3 (100_A to 100_F) are defined, along with a plot of the Wimley-White predicted free energies of transfer to a lipid bilayer interface (black) or to octanol (gray) for each of the mutations.In this study, we sought to examine the role of the chemical nature of residues at the tip of the 2F5 CDR H3 loop in neutralization of HIV-1. Mutations were introduced into the 2F5 CDR H3 loop that altered its hydrophobicity, and the resulting 2F5 mutants were tested both for binding to a gp41 epitope peptide and for neutralization of HIV-1. The results showed that the tip of the 2F5 CDR H3 loop, and specifically its hydrophobic nature, is required for 2F5-mediated neutralization of HIV-1 by means that appear to be independent both of gp41 affinity and of isolate-specific sensitivity to neutralization by 2F5.     

Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01

The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.     

HIV-1 neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes

HIV-1 neutralizing monoclonal antibodies (MAbs) define key targets for vaccine development and are being considered for passive prevention of infection. We analyzed the interaction of MAbs to two independent epitopes on the viral envelope glycoprotein. Potently neutralizing MAbs to the CD4 binding site and V1V2 region displayed no in vitro cross-competition and displayed additive, though not synergistic, neutralization activity. Predicted neutralization coverage of a combination of two MAbs reached 97% on a 208-isolate panel.     

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.