Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralizationpotencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41_int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41_int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41_int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140_CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140_CA018 was higher than that induced by gp41_int-Cys, the majority of animals immunized with gp41_int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.     

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR–NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.     

Binding of HIV-1 gp41-Directed Neutralizing and Non-Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.     

Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41

The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.     

Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41

Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35CCG-N13, N34CCG (a smaller version of N35CCG-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC50 values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6 microg/ml to 60 microg/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.     

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.     

Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41

Background

The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.

Results

Our data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.

Conclusions

Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.     

Identification of the HIV-1 gp41 core-binding motif--HXXNPF

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.     

HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.     

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Steric accessibility of the HIV-1 gp41 N-trimer region

During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

HIV-1跨膜蛋白gp41的截短及表达

将HIV-1跨膜蛋白gp41进行截短,在大肠杆菌中进行表达并纯化。PCR扩增gp41的部分编码基因,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用EcoRI和Sal I切下目的基因,并构建到表达载体pGEX-4T3上,导入宿主细胞BL21(DE3),用IPTG诱导表达,表达产物用亲和层析进行纯化并作相应鉴定。截短的HIV-1跨膜蛋白gp41能直接在大肠杆菌内进行表达,利用亲和层析能方便地将目的蛋白进行纯化,为跨膜蛋白的进一步应用打下基础。     

Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.     

Neutralizing epitopes in the membrane-proximal external region of HIV-1 gp41 are influenced by the transmembrane domain and the plasma membrane

Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure.     

HIV-1 gp41 core with exposed membrane-proximal external region inducing broad HIV-1 neutralizing antibodies

The membrane-proximal external region (MPER) of the HIV-1 gp41 consists of epitopes for the broadly cross-neutralizing monoclonal antibodies 2F5 and 4E10. However, antigens containing the linear sequence of these epitopes are unable to elicit potent and broad neutralizing antibody responses in vaccinated hosts, possibly because of inappropriate conformation of these epitopes. Here we designed a recombinant antigen, designated NCM, which comprises the N- and C-terminal heptad repeats that can form a six-helix bundle (6HB) core and the MPER domain of gp41. Two mutations (T569A and I675V) previously reported to expose the neutralization epitopes were introduced into NCM to generate mutants named NCM(TA), NCM(IV), and NCM(TAIV). Our results showed that NCM and its mutants could react with antibodies specific for 6HB and MPER of gp41, suggesting that these antigens are in the form of a trimer of heterodimer (i.e., 6HB) with three exposed MPER tails. Antigen with double mutations, NCM(TAIV), elicited much stronger antibody response in rabbits than immunogens with single mutation, NCM(TA) and NCM(IV), or no mutation, NCM. The purified MPER-specific antibodies induced by NCM(TAIV) exhibited broad neutralizing activity, while the purified 6HB-specific antibodies showed no detectable neutralizing activity. Our recombinant antigen design supported by an investigation of its underlying molecular mechanisms provides a strong scientific platform for the discovery of a gp41 MPER-based AIDS vaccine.