Structural Details of HIV-1 Recognition by the Broadly Neutralizing Monoclonal Antibody 2F5: Epitope Conformation, Antigen-Recognition Loop Mobility, and Anion-Binding Site

105

135

A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M r 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.membrane-proximal ectodomain region ͉ neutralizing antibody ͉ vaccine design ͉ AIDS ͉ fusion T he 120-nm-diameter HIV-1 is a retrovirus that has a small genome consisting of 9 genes. The enveloped virion surface expresses the trimeric glycoprotein of M r 160,000 (gp160) spike protein, whose gp120 and gp41 subunits are assembled into noncovalently associated heterodimers. Unlike gp120, each gp41 subunit has a transmembrane segment (TM) that inserts into the membrane of the virus. The membrane proximal ectodomain region (MPER) links this TM to the folded gp41 ectodomain. Entry of HIV-1 into human T cells is mediated by attachment of the gp120 subunit to receptor CD4 and then binding to the coreceptor (CCR5 or CXCR4) (1). These interactions result in structural rearrangement of the gp41 subunit, followed by fusion of viral and host cell membranes (2, 3). Thus, antibody-mediated protection against HIV-1 must target the gp120/gp41 envelope trimer, the only viral protein exposed on the virion surface.Although high-titer, strain-specific neutralizing antibodies are readily generated during the course of natural infection or against subunit vaccines, broadly neutralizing antibodies (BNAbs), in contrast, are rarely produced (reviewed in ref. 4). Sequence variability, extensive glycosylation, and immunodominance of those exposed, largely variable segments subvert immune res...

Section: Immersion Depth Changes Of Mper Residues Upon 2f5 and Z13e1mentioning

confidence: 83%

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Song¹,

Sun²,

Coleman³

et al. 2009

105

135

“…These core epitopes were determined by peptide mapping; however, alanine-scanning mutagenesis of the MPER in intact viruses established that only a few residues (D664/K665/W666, W672/F673/W680, and N671/ D674 HXB2 numbering throughout) are essential for 2F5, 4E10, and Z13e1 neutralization, respectively (7,9). Crystal structures of Fab 2F5, 4E10, and Z13e1 complexed with their respective epitope peptides subsequently confirmed that residues DKW, WFW, and ND are buried in the paratopes of these antibodies (10)(11)(12)(13)(14).…”

mentioning

confidence: 93%

“…A similar study was not implemented for 2F5, because the case for 2F5-lipid interactions is less clear, its very occurrence a topic of current debate (21,27,31). Moreover, substitutions of hydrophobic residues at the apex of the 2F5 CDRH3 have been shown to affect antigen binding (32), which may relate to the proposed ability of 2F5 to form contacts with another region of Env on intact trimers (i.e., a region near the fusion peptide of gp41) (11,33).…”

mentioning

confidence: 99%

Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions

Scherer

Leaman

Zwick

et al. 2010

107

118

The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(−) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37-to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-like neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens.4E10 | gp41 | HIV | neutralizing antibody | tryptophan

“…Here we describe a platform for the elicitation of structure-specific antibodies-the epitope-scaffold platform-in which structural mimics of viral neutralizing determinants are grafted into heterologous protein scaffolds using techniques of computational protein design. As a test system, we chose the 2F5 antibody (5, 6), which recognizes an epitope in the membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein, for which we and others have determined a number of atomiclevel structures (7)(8)(9)(10)(11)(12)(13)(14). Although recognition by 2F5 involves not only the structure-specific binding of a gp41 epitope but also nonspecific interactions with membrane (13,(15)(16)(17), the system was nonetheless attractive because of the conformational diversity of the MPER, its extensive structural characterization, and the linear nature of the epitope.…”

mentioning

confidence: 99%

Elicitation of structure-specific antibodies by epitope scaffolds

Ofek

Guenaga

Schief

et al. 2010

271

339

Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structurespecific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant "2F5-epitope scaffolds" possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p < 0.04), while antibody responses against the graft-as dissected residue-by-residue with alanine substitutions-resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation.computational design | epitope transplantation | structural mimicry M onoclonal antibodies of enormous utility have been identified, revolutionizing treatments for autoimmune disorders, infectious disease, and different types of cancers (reviewed in ref. 1). Requirements for nonoral means of delivery and in some contexts prolonged treatment regimens, however, have limited their use. While vaccine modalities have potential for improvements, no clear path exists from a clinically useful monoclonal antibody to elicitation of similar antibodies in a vaccine context. One potential solution is precise immunogen design. The ability of structural biology to provide atomic-level definition of antibodyantigen interactions and of computational biology to manipulate protein structure has raised the possibility-at least for protein antigens-of precisely replicating the antigenic surface recognized by a target antibody. We hypothesized that appropriate immunization with such an antigenic mimic might succeed in eliciting replicas of the original target antibody.As a first step toward solving the vaccine problem of "reelicitation," we undertook the challenge of structure-specific elicitation-the elicitation of antibodies capable of binding the sequence and of inducing the structure of...