Gp41 Membrane Proximal Region Research Articles

Structure, membrane topology and influence of cholesterol of the membrane proximal region: transmembrane helical anchor sequence of gp41 from HIV

During the first steps of HIV infection the Env subunit gp41 is thought to establish contact between the membranes and to be the main driver of fusion. Here we investigated in liquid crystalline membranes the structure and cholesterol recognition of constructs made of a gp41 external region carrying a cholesterol recognition amino acid consensus (CRAC) motif and a hydrophobic membrane anchoring sequence. CD- und ATR-FTIR spectroscopies indicate that the constructs adopt a high degree of helical secondary structure in membrane environments. Furthermore, 15N and 2H solid-state NMR spectra of gp41 polypeptides reconstituted into uniaxially oriented bilayers agree with the CRAC domain being an extension of the transmembrane helix. Upon addition of cholesterol the CRAC NMR spectra remain largely unaffected when being associated with the native gp41 transmembrane sequence but its topology changes when anchored in the membrane by a hydrophobic model sequence. The 2H solid-state NMR spectra of deuterated cholesterol are indicative of a stronger influence of the model sequence on this lipid when compared to the native gp41 sequence. These observations are suggestive of a strong coupling between the transmembrane and the membrane proximal region of gp41 possibly enforced by oligomerization of the transmembrane helical region.

HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism.

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross-reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VHDJH and VLJL knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicry by HIV-1 Env that promotes the evasion of host anti-HIV-1 Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Env gp41 region is a key and safe target for HIV-1 vaccine development.

Increased HIV-1 sensitivity to neutralizing antibodies by mutations in the Env V3-coding region for resistance to CXCR4 antagonists.

HIV-1 passage in cell culture in the presence of chemokine receptor antagonists can result in selection of viruses with env mutations that confer resistance to these inhibitors. In the present study, we examined the effect of HIV-1env mutations that confer resistance to CXCR4 antagonists on envelope (Env) sensitivity to neutralizing antibodies (NAbs). Serial passage of CXCR4-tropic HIV-1 NL4-3 in PM1/CCR5 cells under CXCR4 antagonists KRH-3955, AMD3100 and AMD070 yielded two KRH-3955-resistant, one AMD3100-resistant and one AMD070-resistant viruses. These viruses had multiple env mutations including the Env gp120 V3 region. The majority of viruses having these CXCR4 antagonist-resistant Envs showed higher sensitivity to NAbs 447-52D, b12 and 2F5 targeting the V3 region, the gp120 CD4-binding site and the gp41 membrane proximal region, respectively, compared to NL4-3 WT virus. Recombinant NL4-3 viruses with the V3-coding region replaced with those derived from the CXCR4 antagonist-resistant viruses showed increased sensitivity to NAbs b12, 2F5 and 447-52D. Molecular dynamics simulations of Env gp120 outer domains predicted that the V3 mutations increased levels of fluctuations at the tip and stem of the V3 loop. These results indicate that mutations in the V3-coding region that result in loss of viral sensitivity to CXCR4 antagonists increase viral sensitivity to NAbs, providing insights into our understanding of the interplay of viral Env accessibility to chemokine receptors and sensitivity to NAbs.

Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women.

Broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the HIV envelope glycoprotein have been identified in blood from HIV-1 infected women. We investigated whether antibodies in the genital tract from these women share similar epitope specificities and functional profiles as those in blood. Immunoglobulin (Ig)G and IgA antibodies were isolated from cervicovaginal lavages or Softcups from 13 HIV-infected women in the CAPRISA cohort using Protein G and Peptide M, respectively. Binding antibodies to envelope antigens were quantified by ELISA and binding antibody multiplex assay. Neutralizing antibody titers and epitope targets were measured using the TZM-bl assay with Env-pseudotyped wild-type and mutated viruses. HIV-specific IgG, but not IgA, was detected in genital secretions and the ratio of total IgG to HIV-specific IgG was similar to plasma. HIV-specific IgG reacted with multiple envelope antigens, including V1V2, gp120, gp140 and gp41. Two women had high plasma titers of HIV-specific IgG3 which was also detected in their genital tract samples. IgG from the genital tract had neutralizing activity against both Tier 1 and Tier 2 primary HIV-isolates. Antibodies targeting well known glycan epitopes and the membrane proximal region of gp41 were detected in genital secretions, and matched specificities in plasma. Women with plasma bNAbs have overlapping specificities in their genital secretions, indicating that these predominantly IgG isotype antibodies may transudate from blood to the genital tract. These data provide evidence that induction of systemic HIV-specific bNAbs can lead to antiviral immunity at the portal of entry.

Reactivation of Neutralized HIV-1 by Dendritic Cells Is Dependent on the Epitope Bound by the Antibody

Ab-neutralized HIV-1 can be captured by dendritic cells (DCs), which subsequently transfer infectious HIV-1 to susceptible CD4(+) T cells. In this study, we examined the capacity of early Abs, as well as recently identified broadly neutralizing Abs (bNAbs) targeting different envelope glycoprotein (Env) epitopes, to block HIV-1 transmission by immature and mature DCs to HIV-1-sensitive cells. Three bNAbs directed against the gp41 membrane proximal region of Env (2F5, 4E10, and 10E8) and three gp120 bNAbs targeting the CD4 binding site (b12, VRC01, and NIH45-46) were examined. In addition, eight glycan-dependent bNAbs targeting the V1V2 apex (PG9, PG16, and PGT145), the V3 loop (2G12, PGT121, and PGT128), and the gp120-gp41 interface of Env (PGT151 and 35O22) were tested. bNAbs that bound specific glycans showed, depending on the immature or mature state of the DC, diverse efficiencies in HIV-1 trans-infection. All bNAbs that bound the CD4 binding site blocked trans-infection, whereas all bNAbs directed against the membrane proximal region lost neutralizing activity after DC-mediated HIV-1 transmission. To understand how preneutralized HIV-1 can be transferred as infectious virus by DCs, we followed the processing of 2F5-treated HIV-1 by DCs with confocal microscopy. Inhibition of DC-internalization pathways could not reverse the dissociation of 2F5 from HIV-1, suggesting that Ab dissociation occurs directly at the plasma membrane. Collectively, these findings imply that the location of the epitope and the neutralization capacity of these Abs determine the efficiency of DC-mediated HIV-1 transfer.

Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design.

Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses.

Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1.

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

Generation of HIV-1 potent and broad neutralizing antibodies by immunization with postfusion HR1/HR2 complex

The envelope glycoproteins are major targets for HIV vaccines. The N-terminal and the C-terminal regions of the gp41 interact to form six helix bundles that are responsible for the fusion between the viral and the target cell membranes. Monoclonal antibodies (Abs) able to disrupt the formation of this complex or to interfere with it could inhibit HIV fusion. Most of the well described gp41-specific broadly neutralizing Abs target conformational epitopes within the membrane proximal region of gp41 (MPER) and recognize linear peptides. In this study, a stable human transfected cell line, expressing a well folded heptad repeat regions 1 (HR1)/HR2 postfusion complex was developed. Transfected cells were highly immunogenic in mice and allowed the generation of 40 complex specific B-cell clones. Three of them were able to neutralize efficiently both HIV-1 laboratory adapted virus and primary isolates from different clades. Two neutralizing Abs (Nabs) FC-2 and FC-3 bound to a recombinant folded gp140 and blocked with a high potency HR1/HR2 fusion complex formation in vitro. The conformational epitopes of the three antibodies are different to 2F5, 4E10, D5 or NC-1 and mainly located in the MPER region. Abs were capable of inhibiting syncytium formation by blocking spatial interactions between HR1 and HR2 regions. These findings suggest that immunogenicity of gp41 is achievable and that the use of a fusion complex expressing human cell line is a highly potent immunogen to generate neutralizing antibodies against gp41 envelope glycoprotein.

Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting.

Broadly neutralizing antibodies are not commonly produced in HIV-1 infected individuals nor by experimental HIV-1 vaccines. When these antibodies do occur, it is important to be able to isolate and characterize them to provide clues for vaccine design. CAP206 is a South African subtype C HIV-1-infected individual previously shown to have broadly neutralizing plasma antibodies targeting the envelope gp41 distal membrane proximal external region (MPER). We have now used a fluoresceinated peptide tetramer antigen with specific cell sorting to isolate a human neutralizing monoclonal antibody (mAb) against the HIV-1 envelope gp41 MPER. The isolated recombinant mAb, CAP206-CH12, utilized a portion of the distal MPER (HXB2 amino acid residues, 673–680) and neutralized a subset of HIV-1 pseudoviruses sensitive to CAP206 plasma antibodies. Interestingly, this mAb was polyreactive and used the same germ-line variable heavy (VH1-69) and variable kappa light chain (VK3-20) gene families as the prototype broadly neutralizing anti-MPER mAb, 4E10 (residues 672–680). These data indicate that there are multiple immunogenic targets in the C-terminus of the MPER of HIV-1 gp41 envelope and suggests that gp41 neutralizing epitopes may interact with a restricted set of naive B cells during HIV-1 infection.

Challenges of antibody-mediated protection against HIV-1

Enthusiasm for a role of antibodies in protection against HIV-1 has waxed and waned over the 25-year search for an AIDS vaccine. However, it is now clear that antibodies not only contribute to the control of infection once it is established [1,2], as a series of seminal passive immunization studies in nonhuman primates (NHPs) going back almost 20 years show unequivocally that antibodies can prevent infection with model AIDS viruses [3–14]. These latter observations are currently driving an intense effort to identify epitopes recognized by broadly neutralizing monoclonal antibodies (mAbs) to serve as templates for AIDS vaccine design. To this end, a handful of broadly neutralizing mAbs have been successfully vetted in passive immunization studies in NHPs using SHIVs that are model simian immuno-deficiency viruses (SIV) in which the SIV envelope glycoprotein (Env) is replaced by a HIV-1 Env glycoprotein. The Env glycoprotein is the only HIV-1 protein known to be recognized by neutralizing antibodies. These studies show that SHIV infection can be blocked by individual mAbs specific for epitopes associated with distinct regions of the HIV-1 Env protein. These include the CD4-binding site [9,15] and high-mannose oligosaccharides [7,16] of gp120, as well as the membrane proximal region of gp41 [17]. It should be noted that while individual mAbs can be effective, mAb mixtures [7,18] or neutralizing sera [19,20] might be more potent. Collectively, these studies strongly argue that the correct antibodies can mediate sterilizing (i.e., transmission-blocking) immunity to HIV-1 and that an AIDS vaccine must elicit such antibodies to be effective. There are three key challenges to the development of an ‘antibody-based’ vaccine that are potentially solvable with the experimental tools currently in hand.

Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.

Anticardiolipin Antibodies in HIV Infection Are Independently Associated with Antibodies to the Membrane Proximal External Region of gp41 and with Cell‐Associated HIV DNA and Immune Activation

The demonstration of in vitro cardiolipin reactivity with 2 human immunodeficiency virus (HIV)-specific, broadly neutralizing antibodies (2F5 and 4E10) has prompted reevaluation of autoimmune manifestations in HIV infection. We evaluated autoantibodies, particularly anticardiolipin (aCL), in 67 untreated, asymptomatic, HIV-infected individuals with slow progression of HIV disease and their correlation with 2F5-, 4E10-, b12-, and 2G12-like antibodies directed against epitopes involved in broad neutralization, as well as their correlation with immune activation and virological and clinical indicators. Fifty individuals with chronic HIV infection and standard disease progression were control patients. The majority of the study patients with slow progression of HIV disease were men (78%); their median age was 37 years, their median CD4+ cell count was 672 cells/mL, and their median plasma HIV load was 6200 copies/mL. The majority of the control patients were also men (76%), and most (62%) were receiving highly active antiretroviral therapy; their median age was 43 years, their median CD4+ cell count was 202 cells/mL, and their median plasma HIV load was 2265 copies/mL. aCL immunoglobulin G was detected at similar levels in 49% of patients with slow progression of HIV disease and in 58% of control patients. Viral load was positively associated with aCL in both groups (P < .001), independent of CD4+ cell counts. In patients with slow progression of HIV disease, aCL levels were also correlated with plasma HIV load and cell-associated DNA level (r = 0.486 and r = 0.516, respectively; P < .001), with the proportion of activated CD4+ cells, human leukocyte antigen-DR+ cells (r = 0.445; P = or < .001) but not activated CD8+ T cells, and with the level of B cell activation (quantified by soluble CD23; r = 0.354; P = .007). The level of aCL antibodies was associated with the level of antibodies to the membrane proximal region of gp41 (P = .003). aCL is frequently detected in HIV-infected patients, regardless of disease stage, and is strongly linked with the level of viral replication, the level of CD4+ T and B cell activation, and the level of antibodies to the membrane proximal external region of gp41, independent of CD4+ cell deficiency.

Induction of HIV-1 MPR649–684-specific IgA and IgG antibodies in caprine colostrum using a peptide-based vaccine

Induction of antigen-specific antibodies against HIV-1 in colostrum and milk may help prevent breast milk transmission of the virus. A peptide vaccine against the HIV-1 gp41 membrane proximal region (MPR649–684) was evaluated as proof-of-principle in a caprine model. Pregnant Alpine/Saanen goats were immunized with MPR649–684 peptide conjugated to KLH using alum adjuvant. Immunizations were intramuscular, intranasal, and in the supramammary lymph node region. Samples collected after parturition demonstrated the presence of MPR649–684-specific antibodies in colostrum and serum. These results support the concept that a peptide vaccine can effectively induce MPR649–684-specific sIgA and IgG in the colostrum of a lactating species.

Transcytosis-Blocking Abs Elicited by an Oligomeric Immunogen Based on the Membrane Proximal Region of HIV-1 gp41 Target Non-Neutralizing Epitopes

CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent

The peptide of HIV-1 envelope gp41 (a.a 628–683), referred to herein as P5, contains P1, a conserved galactose-specific lectin domain for binding the mucosal HIV-1-receptor, galactosyl ceramide (GalCer), as shown earlier, and a potential calcium-binding site (a.a 628–648). P1 contains contiguous epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, Z13. However, similar neutralizing antibodies could not be raised in animal model using immunogens based on these epitopes. We now show that the structure of both P5 and P1 peptides, as measured by circular dichroism, differs according to their environment: aqueous or lipidic, and as a function of calcium concentration. P5, but not P1, binds to calcium with a low binding affinity constant in the order of 2.5 × 10 4. Calcium binding results in a conformational change of P5, leading in turn to a decrease in affinity for GalCer. Hence, the affinity of the gp41-lectin site for the galactose harbored by the mucosal HIV-1 receptor GalCer is modulated by the peptide secondary and tertiary structure and the local environment. Therefore, definition of the conformation of this novel extended gp41 membrane proximal region, containing the conserved peptide P1 and the Ca 2+ binding site, could help designing an immunogen efficient at inducing neutralizing anti-HIV-1 antibodies.

Identification of HIV gp41 membrane proximal epitope tetramer reactive populations of B220+ B cells and B220lo/neg basophils in gp41 peptide-immunized mice (45.3)

Abstract 2F5 is a rare, neutralizing human mAb specific for the HIV gp41 membrane proximal region (MPER). Recently, we showed that 2F5 is a cardiolipin-binding, polyreactive autoantibody. To determine if MPER antibodies (Ab) are controlled by tolerance mechanisms, BALB/c mice were immunized with HIV-1 gp140 envelope or gp41 peptides. Immunized mice were screened for serum MPER Ab and for 2F5 antigen-specific B cells using 2F5 epitope B cell tetramers. Immunization in Ribi adjuvant did not induce anti-MPER Ab, whereas similar immunizations in TLR9, an adjuvant designed to break tolerance, did. Interestingly, in mice immunized with gp41 peptide, but not gp140, we found two distinct populations of 2F5 tetramer+ cells in bone marrow, spleen, and blood: a polyclonally-induced B220hi B-cell population that bound 2F5 tetramers via their surface Ig and a B220lo/neg cell population induced by immunization. Histological and cell surface phenotypic analysis of the B220lo/neg 2F5 tetramer+ cells indicated these were primarily basophils that bound 2F5 tetramers via MPER Ab bound to FcR. At present, the immunoregulatory significance of these distinct anti-MPER reactive populations is unknown, but their further characterization and the types of Ab they produce/bind should help in delineating why induction of neutralizing anti-MPER Ab is not achieved, despite the generation of a robust non-neutralizing MPER B cell and Ab response.

The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.

Structural Basis of Enhanced Binding of Extended and Helically Constrained Peptide Epitopes of the Broadly Neutralizing HIV-1 Antibody 4E10

Potent, broadly HIV-1 neutralizing antibodies (nAbs) may be invaluable for the design of an AIDS vaccine. 4E10 is the broadest HIV-1 nAb known to date and recognizes a contiguous and highly conserved helical epitope in the membrane-proximal region of gp41. The 4E10 epitope is thus an excellent target for vaccine design as it is also highly amenable to peptide engineering to enhance its helical character. To investigate the structural effect of both increasing the peptide length and of introducing helix-promoting constraints in the 4E10 epitope, we have determined crystal structures of Fab 4E10 bound to an optimized peptide epitope (NWFDITNWLWYIKKKK-NH 2), an Aib-constrained peptide epitope (NWFDITNAibLWRR-NH 2), and a thioether-linked peptide (NWF CITOWLWKKKK-NH 2) to resolutions of 1.7 Å, 2.1 Å, and 2.2 Å, respectively. The thioether-linked peptide is the first reported structure of a cyclic tethered helical peptide bound to an antibody. The introduced helix constraints limit the conformational flexibility of the peptides without affecting interactions with 4E10. The substantial increase in affinity (10 nM versus 10 4 nM of the IC 50 of the original KGND peptide template) is largely realized by 4E10 interaction with an additional helical turn at the peptide C terminus that includes Leu679 and Trp680. Thus, the core 4E10 epitope was extended and modified to a WFX(I/L)(T/S)XX(L/I)W motif, where X does not play a major role in 4E10 binding and can be used to introduce helical-promoting constraints in the peptide epitope.

Broadly Neutralizing Anti-HIV Antibody 4E10 Recognizes a Helical Conformation of a Highly Conserved Fusion-Associated Motif in gp41

Broadly neutralizing monoclonal antibodies to HIV-1 are rare but invaluable for vaccine design. 4E10 is the broadest neutralizing antibody known and recognizes a contiguous and highly conserved epitope in the membrane-proximal region of gp41. The crystal structure of Fab 4E10 was determined at 2.2 A resolution in complex with a 13-residue peptide containing the gp41 core epitope (NWFDIT). The bound peptide adopts a helical conformation in which the key contact residues, TrpP672, PheP673, IleP675, and ThrP676, map to one face of the helix. The peptide binds in a hydrophobic pocket that may emulate its potential interaction with the host cell membrane. The long CDR H3 of the antibody extends beyond the bound peptide in an orientation that suggests that its apex could contact the viral membrane when 4E10 is bound to its membrane-proximal epitope. These structural insights should assist in the design of immunogens to elicit 4E10-like neutralizing responses.

Two Interaction Modes of the gp41-Derived Peptides with gp41 and Their Correlation with Antimembrane Fusion Activity

Peptides derived from gp41 effectively block the gp41-mediated cell fusion or HIV infection. A 36-mer (naDP178), 51-mer (C51) and 27-mer peptide (C27) from the membrane proximal region of gp41 have been examined their interaction modes with the coiled-coil motif of gp41 presented in thioredoxin (Trx-N) or the bacterially expressed ectodomain of gp41 (Ec-gp41ec). All of these peptides effectively inhibited the gp41-mediated membrane fusion, however, they showed distinct interaction modes with Ec-gp41ec or Trx-N. C51 peptide bound tightly to Trx-N, and it increased the solubility of Ec-gp41ec. naDP178 showed very weak binding affinity to Trx-N, however, it effectively solubilized Ec-gp41ec. In contrast, C27 peptide showed significant binding to Trx-N; however, it did not affect the solubility of Ec-gp41ec. These interaction modes of C-peptides were assumed to be related to their different inhibitory mechanism against gp41-mediated cell fusion.