Oligomeric and conformational properties of a proteolytically mature,disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1(JR-FL) SOS gp140, proteolytically uncleaved gp140 (gp140(UNC)), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140(UNC), SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140(UNC) displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41(ECTO)) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140(UNC) comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140(UNC) were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140(UNC)) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.     

Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.     

Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints.

The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5.     

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.     

Crystal structure of the complex between the F(ab)' fragment of the cross-neutralizing anti-HIV-1 antibody 2F5 and the F(ab) fragment of its anti-idiotypic antibody 3H6

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I β-turn centered on gp41 residues ⁶⁶⁴DKW⁶⁶⁶; in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F_ab and 2F5 F_ab′. Ab2/3H6 F_ab binds to 2F5 F_ab′ via a helix-like protrusion formed by residues ^58(H)RYSPSLNTRL^67(H) of the 2F5 F_ab′ variable domain and proximal to but not overlapping with the gp41 ⁶⁶⁴DKW⁶⁶⁶ epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2γ class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 ⁶⁶²ELDKWAS⁶⁶⁸ epitope.     

Induction of antibodies in rhesus macaques that recognize a fusion-intermediate conformation of HIV-1 gp41

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope ⁶⁶⁴DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope.     

The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site

2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F_ab′ crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an α-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F_ab′-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.     

Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.     

Specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing anti-gp41 2F5 antibody

HIV-1 neutralizing monoclonal antibody (Mab) 2F5 recognizes a membrane-partitioning gp41 sequence. Just recently its capacity to react with cardiolipin has been demonstrated. Here, we have studied the specificity of Mab2F5-phospholipid interactions comparing partitioning into lipid bilayers with recognition of molecular species dispersed in solution. Using a liposome-based ELISA we demonstrate a preferential association with cardiolipin bilayers. When different soluble lysoderivatives were compared in their capacity to inhibit Mab2F5 binding to immobilized HIV-1 peptide epitope, only dilysocardiolipin resulted effective in blocking the process. Dilyso-cardiolipin also competed with native-functional gp41 for 2F5 recognition. Thus, our data support specific cardiolipin recognition by 2F5 that is not dependent on lipid bilayer assembly and involves the epitope-binding site. These findings might be of relevance for understanding the molecular basis of HIV-1 immune evasion.     

In Silico Vaccine Design Based on Molecular Simulations of Rhinovirus Chimeras Presenting HIV-1 gp41 Epitopes

A cluster of promising epitopes for the development of human immunodeficiency virus (HIV) vaccines is located in the membrane-proximal external region (MPER) of the gp41 subunit of the HIV envelope spike structure. The crystal structure of the peptide corresponding to the so-called ELDKWA epitope (HIV-1 HxB2 gp41 residues 662-668), in complex with the corresponding broadly neutralizing human monoclonal antibody 2F5, provides a target for structure-based vaccine design strategies aimed at finding macromolecular carriers that are able to present this MPER-derived epitope with optimal antigenic activity. To this end, a series of replica exchange molecular dynamics computer simulations was conducted to characterize the distributions of conformations of ELDKWA-based epitopes inserted into a rhinovirus carrier and to identify those with the highest fraction of conformations that are able to bind 2F5. The length, hydrophobic character, and precise site of insertion were found to be critical for achieving structural similarity to the target crystal structure. A construct with a high degree of complementarity to the corresponding determinant region of 2F5 was obtained. This construct was employed to build a high-resolution structural model of the complex between the 2F5 antibody and the chimeric human rhinovirus type 14:HIV-1 ELDKWA virus particle. Additional simulations, which were conducted to study the conformational propensities of the ELDKWA region in solution, confirm the hypothesis that the ELDKWA region of gp41 is highly flexible and capable of assuming helical conformations (as in the postfusion helical bundle structure) and β-turn conformations (as in the complex with the 2F5 antibody). These results also suggest that the ELDKWA epitope can be involved in intramolecular—and likely intermolecular—hydrophobic interactions. This tendency offers an explanation for the observation that mutations decreasing the hydrophobic character of the MPER in many cases result in conformational changes that increase the affinity of this region for the 2F5 antibody.     

Cross-reactive HIV-1-neutralizing human monoclonal antibodies identified from a patient with 2F5-like antibodies

The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140(JR-FL). Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW(664-666) core of the 2F5 epitope and two additional upstream residues (L(660,663)). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5--they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.     

Structure-guided Alterations of the gp41-directed HIV-1 Broadly Neutralizing Antibody 2F5 Reveal New Properties Regarding its Neutralizing Function

The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.     

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1(JR-FL), designated HIV-1(JR2), and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LELDKWANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (NWFDISNWLW). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.     

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.     

Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope

The membrane-proximal region of the ectodomain of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the target of three of the five broadly neutralizing anti-HIV-1 antibodies thus far isolated. We have determined crystal structures of the antigen-binding fragment for one of these antibodies, 2F5, in complex with 7-mer, 11-mer, and 17-mer peptides of the gp41 membrane-proximal region, at 2.0-, 2.1-, and 2.2-A resolutions, respectively. The structures reveal an extended gp41 conformation, which stretches over 30 A in length. Contacts are made with five complementarity-determining regions of the antibody as well as with nonpolymorphic regions. Only one exclusive charged face of the gp41 epitope is bound by 2F5, while the nonbound face, which is hydrophobic, may be hidden due to occlusion by other portions of the ectodomain. The structures reveal that the 2F5 antibody is uniquely built to bind to an epitope that is proximal to a membrane surface and in a manner mostly unaffected by large-scale steric hindrance. Biochemical studies with proteoliposomes confirm the importance of lipid membrane and hydrophobic context in the binding of 2F5 as well as in the binding of 4E10, another broadly neutralizing antibody that recognizes the membrane-proximal region of gp41. Based on these structural and biochemical results, immunization strategies for eliciting 2F5- and 4E10-like broadly neutralizing anti-HIV-1 antibodies are proposed.     

Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) complex comprises three gp120 exterior glycoproteins each noncovalently linked to a gp41 transmembrane glycoprotein. Monomeric gp120 proteins can elicit antibodies capable of neutralizing atypically sensitive test viruses in vitro, but these antibodies are ineffective against representative primary isolates and the gp120 vaccines failed to provide protection against HIV-1 transmission in vivo. Alternative approaches to raising neutralizing antibodies are therefore being pursued. Here we report on the antibody responses generated in rabbits against a soluble, cleaved, trimeric form of HIV-1(JR-FL) Env. In this construct, the gp120 and gp41 moieties are covalently linked by an intermolecular disulfide bond (SOS gp140), and an I559P substitution has been added to stabilize gp41-gp41 interactions (SOSIP gp140). We investigated the value of DNA priming and compared the use of membrane-bound and soluble priming antigens and of repeat boosting with soluble and particulate protein antigen. Compared to monomeric gp120, SOSIP gp140 trimers elicited approximately threefold lower titers of anti-gp120 antibodies. Priming with DNA encoding a membrane-bound form of the SOS gp140 protein, followed by several immunizations with soluble SOSIP gp140 trimers, resulted in antibodies capable of neutralizing sensitive strains at high titers. A subset of these sera also neutralized, at lower titers, HIV-1(JR-FL) and some other primary isolates in pseudovirus and/or whole-virus assays. Neutralization of these viruses was immunoglobulin mediated and was predominantly caused by antibodies to gp120 epitopes, but not the V3 region.