Secondary Logo

Journal Logo


Anti-HIV-1 antibodies 2F5 and 4E10 interact differently with lipids to bind their epitopes

Franquelim, Henri Ga; Chiantia, Salvatoreb; Veiga, Ana Saloméa,c; Santos, Nuno Ca; Schwille, Petrab; Castanho, Miguel ARBa

Author Information
doi: 10.1097/QAD.0b013e328342ff11



The viral entry process, mediated by the envelope (Env) gp41–gp120 complex [1–3], represents a major target for HIV-1 inhibition. Due to their conserved structure and specific functions, envelope proteins seem to display promising targets for a potential vaccine development against HIV-1 [4]. So far, few broad neutralizing monoclonal antibodies (mAbs) have been isolated from HIV-1-infected patients [4,5]. 2F5 and 4E10 are two of these and recognize epitopes localized on the gp41 membrane-proximal external region (MPER) [4,5]. MPER is a tryptophan (Trp)-rich conserved region that is located near the viral membrane and seems to have important functions during the membrane fusion process [6,7]. Nevertheless, the efforts to obtain neutralizing antibodies using only MPER epitopes as antigens were proven to be difficult [8,9]. Due to the proximity of MPER to the viral membrane, lipid bilayers may be required for the development of antibodies against this region. In the native gp41, MPER corresponds to the extracellular region directly adjacent to the viral membrane [10] (see Fig. 1a). Peptides corresponding to this region are mainly unstructured in aqueous solution [11]; however, acquire defined α-helical secondary structure within lipid bilayers [12,13]. Recent studies have provided additional insights concerning MPER's structure, demonstrating that this region possesses two helices linked by a kink, with the N-terminal region more exposed to the aqueous medium and the C-terminal region inserted in a shallow position on lipid membranes [14]. Additionally, it was demonstrated that MPER-derived peptides interact both with fluid (liquid disordered – ld) and cholesterol-enriched (liquid ordered – lo) membranes [15,16]. All of this demonstrates the importance of lipid membranes for the orientation and presentation of the MPER epitopes to neutralizing antibodies.

Fig. 1
Fig. 1:
Location of the two epitopes on the membrane proximal external region and the different phases of the experimental design. (a) Illustration of the location of the 2F5 and 4E10 epitopes on the membrane proximal external region (MPER) of HIV-1 gp41. The amino acid sequence corresponds to the MPER peptide used during this work. (b–d) Schematic representations of the different phases of the experimental design, concerning MPER and mAbs order of incubation on the supported lipid bilayers (SLBs). The MPER is represented in two colors: black, 2F5 epitope region; gray, 4E10 epitope region. CT, cytoplasmic domain; FP, fusion peptide; HR, heptad repeats; MPER, membrane proximal external region; TM, transmembrane domain.

2F5 and 4E10 [7,17,18] have been studied as HIV-targeted antiretrovirals [19,20]. These mAbs possess structural adaptations to bind their epitopes in the lipid membrane interface [21–23]. For instance, they possess longer and more hydrophobic third complementarity-determining regions on the heavy chain (CDR H3) [23–25]. Furthermore, it has been reported that these Abs, mostly 4E10, interact with zwitterionic and anionic lipid membranes prior or during epitope recognition [26–39]. Recent studies reported that most of the residues in the CDR H3 region are not participating in epitope recognition, but are essential for membrane binding [11,40–44]. Modifications in some critical residues in the hydrophobic CDR H3 region can decrease lipid binding without affecting significantly the epitope recognition.

Despite the recent developments regarding epitope and membrane affinities, there is still a lot of information to be unraveled regarding 2F5 and 4E10 mode of action at the molecular level. Therefore, the aim of this work is to elucidate the neutralizing mAbs 2F5 and 4E10 molecular interaction with their epitopes at the membrane level. The membrane-binding properties of 2F5 and 4E10 were studied by the use of supported lipid bilayers (SLBs) as biomembrane models, with and without phase separation. Atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM) were used to evaluate the mAbs–membrane interactions, in the presence and absence of a MPER-derived peptide containing both mAbs epitopes. 2F5 is able to dock the MPER peptide on the membrane, whereas 4E10 is more intrusive and extracts the MPER from the lipid bilayer. The results obtained in this work contribute to the understanding of the molecular determinants underneath the differential efficacy of 2F5 and 4E10 targeting the HIV-1 MPER and distinguish their modes of action.

Materials and methods

Sample preparation

MPER (NEQELLELDKWASLWNWFNITNWLWYIK-amide; >95% purity) and Fam-MPER (5,6-carboxyfluorescein-NEQELLELDKWASLWNWFNITNWLWYIK-amide; >92% purity) peptides were obtained from Bachem AG (Bubendorf, Switzerland). mAbs 2F5 and 4E10 were purchased from Polymun Scientific (Vienna, Austria). 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycro-3-phosphocholine (DOPC), chicken egg yolk sphingomyelin and cholesterol (Chol) were from Avanti Polar Lipids (Alabaster, Alabama, USA), whereas cholesteryl-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (FL-Bodipy-Chol) and 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate (TMR-Bodipy-Chol) were obtained from Molecular Probes (Eugene, Oregon, USA). Ten millimolar HEPES buffer pH 7.4, 150 mmol/l NaCl and 3 mmol/l NaN3 was used throughout the studies.

MPER and Fam-MPER stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted to desired concentrations with HEPES buffer. In order not to perturb the lipid organization [45], DMSO concentration was maintained below 0.14% (v/v) in the peptide samples throughout the experiments. 2F5 and 4E10 stock solutions (11 mg/ml) were stored in 2 mmol/l acetate buffer with 10% maltose and diluted to the desired concentrations using the HEPES buffer.

SLBs were prepared by deposition on freshly cleaved mica, in the presence of 1 mmol/l CaCl2, of POPC or DOPC: sphingomyelin: Chol (3: 3: 2) small unilamellar vesicles obtained by sonication, as described in [46]. FL-Bodipy-Chol and TMR-Bodipy-Chol molar percentages in the lipid membranes were kept below 0.005 and 0.01% for POPC and DOPC: sphingomyelin: Chol (3: 3: 2), respectively.

Instrumental setup

AFM measurements were performed on a JPK Instruments Nanowizard BioAFM (Berlin, Germany) mounted on a Carl Zeiss laser scanning microscope (LSM) Meta 510 system (Jena, Germany) [47]. Intermittent contact imaging was performed using uncoated silicon cantilevers CSC38 from MikroMasch (Tallinn, Estonia) with typical spring constants of 0.01–0.2 N/m. The scan rate was set to less than 1 Hz and the cantilever oscillation frequency between 10 and 20 kHz. The force applied on the sample was minimized by continuously adjusting the set point and gain during the imaging.

CLSM and line-scan fluorescence correlation spectroscopy (FCS) measurements were performed on the LSM Meta 510 system as described elsewhere [48]. Highly sensitive fluorescence confocal microscopy measurements were conducted using a commercial Carl Zeiss ConfoCor3 system (Jena, Germany) with avalanche photodiode (APD) detectors. The 488-nm line of the argon-ion laser (to excite Fam-MPER and FL-Bodipy-Chol), the 543 nm helium–neon laser (to excite TMR-Bodipy-Chol) and a 40 × NA 1.2 UV–VIS–IR C Apochromat water-immersion objective were used in both setups. CLSM images were acquired with a 512 pixel × 512 pixel resolution and typical 6.4 μs per pixel scanning rate.

Experimental design

For all AFM and CLSM studies, the interactions of 2F5 or 4E10 mAbs (0.3–300 nmol/l) and MPER/Fam-MPER peptides (0.2–500 nmol/l) with POPC or DOPC: sphingomyelin: Chol (3: 3: 2) SLB (containing Bodipy-Chol) were followed upon successive additions of small volumes of stock solutions to the desired concentrations. The samples were allowed to incubate for 15 min at room temperature after each addition. Controls, with buffer instead of mAbs or MPER, were performed. When two components (mAbs and peptides) were being evaluated, the experiments were carried out as follows (see also Fig. 1b–d). Whenever a component B (mAbs or MPER, respectively) was added to membranes in which a component A (MPER or mAbs, respectively) was already present, the excess of free component A in solution was always rinsed with buffer to ensure the interaction of only component B with the membrane-bound component A. Images were obtained at least for three to five regions of the lipid bilayers. Statistical analysis was performed using repeated or one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (interval of confidence 95%).


Confocal and atomic force microscopy studies

Interaction of the antibodies with lipid bilayers in the absence of the peptide epitopes

The interaction of both 2F5 and 4E10 (0.3–300 nmol/l) with SLB composed by POPC or the ternary mixture DOPC: sphingomyelin: Chol (3: 3: 2) deposited on freshly cleaved mica were analyzed by AFM and CLSM. POPC was used to mimic the ld bulk of an eukaryotic plasma membrane, whereas the DOPC: sphingomyelin: Chol mixture was used to mimic the coexistence of a ld phase with lo domains, enriched in Chol and sphingomyelin, usually termed lipid rafts [49,50]. The HIV-1 membrane that constitutes the enveloped is highly ordered and rich in cholesterol [51]. Using AFM and CLSM, we can easily distinguish both ld and lo domains on SLB, as seen in Fig. 2a and d (see legend for more details).

Fig. 2
Fig. 2:
Atomic force microscopy and confocal laser scanning microscopy of 1,2-dioleyl-sn-glycro-3-phosphocholine: sphingomyelin: Chol-supported lipid bilayers. Atomic force microscopy (AFM) (a–c) and confocal laser scanning microscopy (CLSM) (d–f) images of DOPC: sphingomyelin: Chol supported lipid bilayers on mica in the absence and presence of 100 nmol/l of mAbs 4E10 or 2F5. The brighter domains correspond to the higher lo regions in the AFM images and to the ld regions in the CLSM images, respectively. The mAbs aggregates correspond to the brightest regions in AFM and to the darkest regions in CLSM. Similar results were observed for 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) bilayers and at different mAbs concentrations. DOPC, 1,2-dioleyl-sn-glycro-3-phosphocholine. Scale bar in CLSM images is 10 μm.

In the presence of either 2F5 or 4E10, no extensive modifications in POPC bilayer thickness (data not shown – DNS) nor perturbations on the phase-separated domains of DOPC: sphingomyelin: Chol bilayers occur, as revealed by the AFM results (Fig. 2a–c). Absence of significant changes in domain morphology was also corroborated by CLSM (Fig. 2d–f). Moreover, by APD imaging, no significant modifications of the lipid fluorescence intensities were obtained (DNS). Additional FCS measurements to assess the dynamics of POPC phospholipids in the presence and absence of 2F5 or 4E10 did not demonstrate significant interference of either mAbs on the lipid diffusion coefficient (∼3 μm2/s, in agreement with [48]).

Nevertheless, the mAbs formed lipid-segregated aggregates within the SLB on mica. CLSM reveals that both mAbs form segregated regions with irregular forms and lacking fluorescence signal (Fig. 2e and f), preferentially on the more fluid membrane domains. Using AFM, irregular aggregates on the lipid bilayers were observed (Fig. 2b and c), which are clearly related to the darkest regions observed on the CLSM images. 4E10 aggregates tend to be higher than 2F5 aggregates. Typical heights of 3.2 ± 1.3 and 16.9 ± 6.1 nm were retrieved for 100 nmol/l 2F5 and 4E10, respectively (Fig. S1, supplementary data, As such extensive effects were not observed when buffer (control) or even MPER peptide (see following section) were added, the aggregates here observed can be interpreted as direct consequence of at least a transient interaction and destabilization of intact lipid membranes by 2F5 and 4E10.

Interaction of the antibodies with lipid bilayers with inserted peptide epitopes

The study of 2F5 and 4E10 effects on lipid bilayers with the prebound MPER was performed using an MPER-derived peptide (see Fig. 1a). This peptide (amino acids 656–683 by HXBc2 numbering) overlaps both 2F5 epitope core sequence (ELDKWA, amino acids 662–667) and 4E10 epitope core sequence (NWFNIT, amino acids 671–676) and is extended by 6 amino acid residues at both N and C terminals. This peptide mimics better the regions of gp41 presented to 2F5 and 4E10 compared with the epitope cores alone [27].

Initially, the membrane interaction properties of the nonlabeled MPER-derived peptide were evaluated. CLSM images of POPC (Fig. 3e) and DOPC: sphingomyelin: Chol (DNS) bilayers in the presence of MPER (0.2–200 nmol/l) did not present significant perturbations when compared with the controls (Fig. 3d). Furthermore, line scan FCS measurements confirmed that the peptide (200 nmol/l) did not induce major changes in the lipid dynamics and diffusion coefficients of POPC SLB (DNS). Additionally, AFM experiments did not reveal major interferences of the peptide on the membrane bilayers structure (DNS). Nevertheless, it is worth stressing that prolonged AFM imaging in the presence of MPER peptide could contribute to an increased lipid bilayer perturbation and the formation of holes (as seen in Fig. 3b), which are not observed in the controls (Fig. 3a).

Fig. 3
Fig. 3:
Atomic force microscopy and confocal laser scanning microscopy of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine-supported lipid bilayers. Atomic force microscopy (a–c) and confocal laser scanning microscopy (CLSM) (d–f) images of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) supported lipid bilayers on mica prior and after additions of firstly 200 nmol/l membrane proximal external region (MPER) and secondly 100 nmol/l 4E10 or 2F5. Scale bar in CLSM images is 10 μm.

The addition of 2F5 (DNS) and 4E10 (Fig. 3c and f) to SLB on mica with prebound MPER revealed results similar to those observed for the interaction of both mAbs with the membranes alone (Fig. 3b and e). No major perturbations on the bilayer thickness and phase-separated domains were detected by CLSM or AFM imaging, except the formation of the lipid-segregated aggregates of mAbs, already discussed.

Highly sensitive avalanche photodiode fluorescence microscopy studies

To unravel details on the interplay between mAbs, MPER-derived peptides and lipids, an APD-imaging microscopy approach was utilized, following the fluorescence emission of a 5,6-carboxyfluorescein labeled MPER-derived peptide (Fam-MPER) at the membrane level. It is known that the MPER C terminal is highly enriched in Trp residues, which are essential for the MPER membrane binding [13–15,52]. Therefore, to ensure minimal interference of the probe on the binding of the peptide to membranes, the peptide was labeled at the N terminus.

Fam-MPER demonstrated a preferential interaction toward the more fluid ld membrane domains. As shown in Fig. 4a, the peptide's fluorescence emission is more intense in POPC (ld) and less intense in the overall domains coexisting in DOPC: sphingomyelin: Chol (ld + lo) membranes. Nonetheless, if we only count for the fluorescence intensities on ld of phase-separated bilayers, the intensities are similar to those observed for the peptide interacting with POPC membranes. This indicates that the peptide presents a preferential and facilitated insertion in ld membranes compared with cholesterol-enriched lo domains. It is also to notice that no significant changes on the lipid TMR-Bodipy-Chol fluorescence intensity were reported as the peptide concentration was increased (DNS), indicating an absence of major lipid perturbation or significant lipid removal.

Fig. 4
Fig. 4:
Effect of mAbs 2F5 and 4E10 membrane prebinding on the Fam-membrane proximal external region interaction with supported lipid bilayers. (a) Interaction of Fam-membrane proximal external region (MPER) with 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and DOPC: sphingomyelin: Chol (overall and ld alone) supported lipid bilayer (SLB). (b) Confocal laser scanning microscopy (CLSM) images of mAbs aggregates on POPC bilayers in the presence of 50 nmmol/l Fam-MPER and 100 nmol/l 2F5 and 4E10. The red fluorescence represents the Bodipy–TMR–Chol-labeled bilayers, whereas the green fluorescence represents the Fam-labeled MPER. Scale bar is 5 μm. (c–d) Interaction of Fam-MPER with POPC (c) and DOPC: sphingomyelin: Chol (d) SLB, in the absence (black) or presence of preincubated 100 nmol/l 2F5 (red) and 4E10 (blue). In (a), (c) and (d), the interactions were assessed by the variation in Fam-MPER fluorescence intensity on 15 μm × 15 μm regions. Error bars correspond to the standard deviation of five to seven measurements on separated regions of the bilayers. DOPC, 1,2-dioleyl-sn-glycro-3-phosphocholine.

After confirming the interaction of Fam-MPER with POPC and DOPC: sphingomyelin: Chol SLB (Fig. 4a), we assessed the effect of 2F5 and 4E10 on the binding of peptide to membranes, as illustrated in Fig. 1b–d. Fam-MPER fluorescence co-localizes within the lipid-segregated mAbs aggregates on the surface of lipid bilayers (Fig. 4b). This is an unequivocal demonstration of the binding of both 4E10 and 2F5 to their epitopes.

Interaction of the peptide epitopes with lipid bilayers preincubated with antibodies

As membrane binding of both 2F5 and 4E10 has been proposed [4,34], we analyzed how a mAbs preincubation at the bilayer level would affect MPER binding (Fig. 1b). Preincubation of 2F5 and 4E10 on the SLB indeed interfered with the MPER membrane binding. Both mAbs decreased the binding of the peptide to POPC (Fig. 4c) and DOPC: sphingomyelin: Chol (Fig. 4d) systems. This decrease may be related to the capture of the peptide by the mAbs still in solution and/or to a weaker interaction of the MPER–mAbs complex with membranes, when compared with MPER alone. It has been reported that 2F5 does not bind or binds less to membranes than 4E10 [4,27]. The higher decrease in the peptide fluorescence at the bilayer level caused by 2F5, in comparison with 4E10, can be a consequence of this differential binding extent. 4E10 would have more affinity to the membrane, promoting therefore more MPER binding compared with 2F5.

Interaction of the antibodies with lipid bilayers with inserted peptide epitopes

Our second approach consisted in determining the effects of mAbs on MPER already bound to membranes (Fig. 1c). This experimental design is a better mimetic of what happens in a biological setting, where the mAbs binds to the MPER in a lipid environment [27].

As the peptide is in equilibrium between the aqueous-soluble and membrane-bound forms, performing successive additions of mAbs would perturb this equilibrium. This would remove peptide from the lipid bilayer, consequently decreasing the fluorescence intensity comparative to controls. Therefore, we performed successive additions of fixed concentration of mAbs (100 nmol/l) to ensure that the effects of the mAbs binding on the membrane-bound MPER are not masked by this equilibrium shift.

As can be seen in Fig. 5a, for POPC, and in Fig. 5b for DOPC: sphingomyelin: Chol membranes, we observed that 4E10 promoted an extensive decrease in the peptide's fluorescence intensity compared with the control. In contrast, in the presence of 2F5, no reduction in the fluorescence intensity was observed. This indicates that 4E10 promotes the extraction of MPER from the SLB, whereas 2F5 increases the docking of the peptide to the SLB. Furthermore, the addition of either mAbs did not cause any perturbation on the lipid signal and promoted the formation of lipid-segregated complexes (Fig. 5c), as expected.

Fig. 5
Fig. 5:
Effect of mAbs 2F5 and 4E10 on supported lipid bilayers in the presence of membrane proximal external region. (a, b) Perturbations in the fluorescence intensity of prebound Fam-membrane proximal external region (MPER) in (a) 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and (b) DOPC: sphingomyelin: Chol membranes. The columns represent the successive additions of buffer (black), 4E10 (blue) and 2F5 (red). (c) CLSM images of mAbs aggregates on POPC bilayers in the presence of 200 nmol/l Fam-MPER and 100 nmol/l 2F5 or 4E10. The red fluorescence represents the Bodipy–TMR–Chol-labeled bilayers, whereas the green fluorescence represents the Fam-labeled MPER. Scale bar is 10 μm. (d) Binding of MPER–mAbs complexes to POPC supported lipid bilayer (SLB). MPER and mAbs (both at 100 nmol/l) were preincubated before adding to POPC bilayers. (e) Schematic representation of the putative modes of action of 2F5 and 4E10 on planar lipid bilayers in the presence of MPER. The N-terminal domain of MPER is presented in red, whereas the C-terminal domain is in blue. Antibodies are colored in green. DOPC, 1,2-dioleyl-sn-glycro-3-phosphocholine. In panels (a), (b) and (d), interactions were assed via variation of the Fam-MPER fluorescence intensities on 7.5 μm × 7.5 μm lipid membrane regions; error bars correspond to the standard deviation of three to five measurements on separated regions of the bilayers. *P < 0.05, **P < 0.01, ***P < 0.001 vs. fluorescence intensity prior addition of buffer or mAbs, repeated ANOVA, Bonferroni test; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. buffer-treated (a, b) or peptide-treated (d) controls.

Interaction of antibody–epitope complexes with lipid bilayers

To evaluate the lipid-binding properties of mAbs–MPER complexes, 2F5 or 4E10 were preincubated with MPER (Fig. 1d). The mAbs binding to Fam-MPER was confirmed by FCS. An approximate three-fold increase in Fam-MPER diffusion time (τD) was observed (unbound: τD = 43 ± 4 ms; bound to mAb: τD = 118 ± 15 ms), confirming mAbs–epitope binding. The complexes were then allowed to interact with POPC membranes. For both mAbs–MPER preformed complexes (Fig. 5d), a reduction in the peptide's fluorescence intensity at the membrane level was observed, when compared with MPER alone. This result suggests that the mAbs hinder MPER-membrane binding. Furthermore, a more pronounced reduction was observed for 4E10–MPER complexes. This indicates that 4E10–MPER complex has less affinity for membranes, compared with the 2F5–MPER complex, which is in agreement with the previous results.


2F5 and 4E10 are mAbs directed against the HIV-1 gp41 MPER. It has been reported that 2F5 holds a stronger binding toward its ELDKW epitope core compared with 4E10 with its NWF(D/N)IT epitope core [4]. Nevertheless, concerning the membrane binding properties, the interaction of 2F5 is still a matter of debate [4,26]. For 4E10, the interaction with membranes is generally well accepted (reviewed in [4]). Most of the studies agree, however, that both mAbs would bind to MPER in a lipid-bound state [4,13,27], confirming the importance of membranes in their mode of action.

In this study, we evaluated the interaction of those mAbs with lipid membranes and with MPER-derived peptides containing their core epitopes. In terms of the membrane alterations caused by 2F5 and 4E10, we observed from the CLSM and AFM images that these mAbs do not cause significant perturbations on phase separation, lipid dynamics, bilayer thickness or lipid removal. Nevertheless, extensive aggregation of the mAbs, with lipid segregation, was imaged in the presence and absence of MPER. These results show the ability of the mAbs to be intrusive and induce confined local disorder in the membranes. Studies reporting the aggregation of mAbs on cholesterol-enriched membranes containing MPER [16] further illustrate the relevance of this observation, because they show the ability of the mAbs to perturb membranes locally exposing epitopes for interaction and to promote cooperative effects that may facilitate the interaction with gp41 because this protein is in trimeric state in the virus. It is worth stressing at this point that, relatively to the interaction of the MPER-derived peptide with SLB, no significant perturbations were detected. Moreover, using highly sensitive APD imaging, we observed that the peptide prefers ld domains in opposition to the cholesterol-enriched lo domains, which shows its tendency to co-localize in the mAbs-induced aggregates, favoring binding between both.

After evaluating the affinities of mAbs and MPER to lipid bilayers separately and more qualitatively, we were prompted to study more elaborated experimental designs to describe in more biologically accurate settings the mAbs–MPER combined membranes interactions. Using that approach, we could unravel distinct mode of actions for 2F5 and 4E10 on membrane-bound MPER. First of all, by preincubating the mAbs before the addition of the MPER-derived peptide, we retrieved less peptide binding toward membranes, as already described [36,37]. The differences obtained between 2F5 and 4E10 could be explained by the fact that 4E10 has a higher membrane affinity. Therefore, 4E10 would be more concentrated on the membrane, enabling more MPER to bind to membranes, in opposition to 2F5 [27,37]. Our second approach consisted in analyzing the effects of 2F5 and 4E10 on membrane-bound MPER, which is close to what actually happens on the viral surface. The mAbs presented the same effect either in POPC or DOPC: sphingomyelin: Chol membranes, demonstrating no clear membrane phase binding preferences for 2F5 and 4E10 in the presence of MPER peptide.

Nevertheless, significant differences relative to the mode of action of both mAbs were observed: it was clearly demonstrated that the binding of 2F5 causes peptide docking on the membrane, in opposition to 4E10 that promotes the peptide extraction (Fig. 5e). This may be due to the different in-depth location of both epitopes on the MPER. The ELDKW core (more N terminal) is more exposed to the aqueous medium than the NWF(D/N)IT core (more C terminal), which is more inserted and protected by the lipid bilayer [13,14]. The C-terminal region of MPER is highly hydrophobic and, therefore, crucial for lipid interactions. In contrast to 2F5, binding of 4E10 to its epitope hinders interaction of the C-terminal region of MPER to membranes. 2F5 can bind to its epitope at the surface of the membrane; at variance, 4E10 needs to interact more deeply with membranes, promoting the pulling and extraction of its epitope from an in-depth localization (Fig. 5e). In agreement, the 2F5–MPER complex has a higher membrane affinity than the 4E10–MPER complex (Fig. 5d). Other studies also reported different behaviors of 2F5 and 4E10 at the membrane level, supporting our observations [14,27,44,53,54]. For instance, in SPR experiments, higher dissociation rate constants were reported for 4E10 interacting with membranes in the presence of its epitope, in contrast to 2F5 where dissociation is almost irrelevant.

The results obtained in this work provide insights into the mode of action of 2F5 and 4E10 at the membrane level and are a valuable contribution to the understanding of their different biological efficacies. Although more efficient epitope binding for 2F5 was acknowledged [4] and lipid preferential reactivity was reported for 4E10 [34], the molecular reasons underlying these phenomena have so far remained uncertain. Those differences are related not only to the direct interaction with the lipids themselves but also with the specific microenvironments of the core epitopes in the lipid bilayer. While the core epitopes of both mAbs are very close in terms of gp41 amino-acid sequence, the short difference between them determines different degrees of exposure in the lipid environment, with 4E10 needing to be more intrusive in the perturbation of the interaction of its core epitope with the membrane lipids [14,53]. This may be the key to its improved neutralizing effect.


The authors are grateful for the support from the Max Planck Gesellschaft (Germany). Fundação para a Ciência e Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (Portugal) is acknowledged for funding (SFRH/BD/39039/2007 grant to H.G.F. and projects PTDC/QUI-BIQ/104787/2008, PTDC/QUI/69937/2006 and REEQ/140/BIO/2005).

Experimental design: All.

Performing experiments: H.G.F. and S.C.

Data discussion: All.

Writing the paper: H.G.F. and M.C.


1. Chan DC, Kim PS. HIV entry and its inhibition. Cell 1998; 93:681–684.
2. Root MJ, Steger HK. HIV-1 gp41 as a target for viral entry inhibition. Curr Pharm Des 2004; 10:1805–1825.
3. Castagna A, Biswas P, Beretta A, Lazzarin A. The appealing story of HIV entry inhibitors: from discovery of biological mechanisms to drug development. Drugs 2005; 65:879–904.
4. Montero M, van Houten NE, Wang X, Scott JK. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol Mol Biol Rev 2008; 72:54–84.
5. Nelson JD, Brunel FM, Jensen R, Crooks ET, Cardoso RM, Wang M, et al. An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J Virol 2007; 81:4033–4043.
6. Muster T, Steindl F, Purtscher M, Trkola A, Klima A, Himmler G, et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol 1993; 67:6642–6647.
7. Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO, Binley JM, et al. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 2001; 75:10892–10905.
8. Bures R, Gaitan A, Zhu T, Graziosi C, McGrath KM, Tartaglia J, et al. Immunization with recombinant canarypox vectors expressing membrane-anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 2000; 16:2019–2035.
9. Gao F, Weaver EA, Lu Z, Li Y, Liao HX, Ma B, et al. Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein. J Virol 2005; 79:1154–1163.
10. Zhu P, Liu J, Bess J Jr, Chertova E, Lifson JD, Grise H, et al. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 2006; 441:847–852.
11. Alam SM, Morelli M, Dennison SM, Liao HX, Zhang R, Xia SM, et al. Role of HIV membrane in neutralization by two broadly neutralizing antibodies. Proc Natl Acad Sci U S A 2009; 106:20234–20239.
12. Suarez T, Nir S, Goni FM, Saez-Cirion A, Nieva JL. The pretransmembrane region of the human immunodeficiency virus type-1 glycoprotein: a novel fusogenic sequence. FEBS Lett 2000; 477:145–149.
13. Lorizate M, Huarte N, Saez-Cirion A, Nieva JL. Interfacial pretransmembrane domains in viral proteins promoting membrane fusion and fission. Biochim Biophys Acta 2008; 1778:1624–1639.
14. Sun ZY, Oh KJ, Kim M, Yu J, Brusic V, Song L, et al. HIV-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane. Immunity 2008; 28:52–63.
15. Veiga AS, Castanho MA. The influence of cholesterol on the interaction of HIV gp41 membrane proximal region-derived peptides with lipid bilayers. FEBS J 2007; 274:5096–5104.
16. Saez-Cirion A, Nir S, Lorizate M, Agirre A, Cruz A, Perez-Gil J, Nieva JL. Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring. J Biol Chem 2002; 277:21776–21785.
17. Trkola A, Pomales AB, Yuan H, Korber B, Maddon PJ, Allaway GP, et al. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J Virol 1995; 69:6609–6617.
18. Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, Steindl F, Katinger H. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 2001; 17:1757–1765.
19. Julg B, Goebel FD. What's new in HIV/AIDS? Neutralizing HIV antibodies: do they really protect? Infection 2005; 33:405–407.
20. Vcelar B, Stiegler G, Wolf HM, Muntean W, Leschnik B, Mehandru S, et al. Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data. AIDS 2007; 21:2161–2170.
21. Grundner C, Mirzabekov T, Sodroski J, Wyatt R. Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins. J Virol 2002; 76:3511–3521.
22. Lenz O, Dittmar MT, Wagner A, Ferko B, Vorauer-Uhl K, Stiegler G, Weissenhorn W. Trimeric membrane-anchored gp41 inhibits HIV membrane fusion. J Biol Chem 2005; 280:4095–4101.
23. Ofek G, Tang M, Sambor A, Katinger H, Mascola JR, Wyatt R, Kwong PD. Structure and mechanistic analysis of the antihuman immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J Virol 2004; 78:10724–10737.
24. Cardoso RM, Zwick MB, Stanfield RL, Kunert R, Binley JM, Katinger H, et al. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 2005; 22:163–173.
25. Julien JP, Bryson S, Nieva JL, Pai EF. Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site. J Mol Biol 2008; 384:377–392.
26. Veiga AS, Castanho MA. The membranes' role in the HIV-1 neutralizing monoclonal antibody 2F5 mode of action needs re-evaluation. Antiviral Res 2006; 71:69–72.
27. Veiga AS, Pattenden LK, Fletcher JM, Castanho MA, Aguilar MI. Interactions of HIV-1 antibodies 2F5 and 4E10 with a gp41 epitope prebound to host and viral membrane model systems. Chembiochem 2009; 10:1032–1044.
28. Haynes BF, Fleming J, St Clair EW, Katinger H, Stiegler G, Kunert R, et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 2005; 308:1906–1908.
29. Sanchez-Martinez S, Lorizate M, Hermann K, Kunert R, Basanez G, Nieva JL. Specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing antigp41 2F5 antibody. FEBS Lett 2006; 580:2395–2399.
30. Sanchez-Martinez S, Lorizate M, Katinger H, Kunert R, Nieva JL. Membrane association and epitope recognition by HIV-1 neutralizing antigp41 2F5 and 4E10 antibodies. AIDS Res Hum Retroviruses 2006; 22:998–1006.
31. Alam SM, McAdams M, Boren D, Rak M, Scearce RM, Gao F, et al. 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. J Immunol 2007; 178:4424–4435.
32. Beck Z, Karasavvas N, Tong J, Matyas GR, Rao M, Alving CR. Calcium modulation of monoclonal antibody binding to phosphatidylinositol phosphate. Biochem Biophys Res Commun 2007; 354:747–751.
33. Brown BK, Karasavvas N, Beck Z, Matyas GR, Birx DL, Polonis VR, Alving CR. Monoclonal antibodies to phosphatidylinositol phosphate neutralize human immunodeficiency virus type 1: role of phosphate-binding subsites. J Virol 2007; 81:2087–2091.
34. Alving CR. 4E10 and 2F5 monoclonal antibodies: binding specificities to phospholipids, tolerance, and clinical safety issues. AIDS 2008; 22:649–651.
35. Beck Z, Karasavvas N, Matyas GR, Alving CR. Membrane-specific antibodies induced by liposomes can simultaneously bind to HIV-1 protein, peptide, and membrane lipid epitopes. J Drug Target 2008; 16:535–542.
36. Huarte N, Lorizate M, Kunert R, Nieva JL. Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies. FEBS Lett 2008; 582:3798–3804.
37. Huarte N, Lorizate M, Maeso R, Kunert R, Arranz R, Valpuesta JM, Nieva JL. The broadly neutralizing antihuman immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5. J Virol 2008; 82:8986–8996.
38. Matyas GR, Beck Z, Karasavvas N, Alving CR. Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1. Biochim Biophys Acta 2009; 1788:660–665.
39. Matyas GR, Wieczorek L, Beck Z, Ochsenbauer-Jambor C, Kappes JC, Michael NL, et al. Neutralizing antibodies induced by liposomal HIV-1 glycoprotein 41 peptide simultaneously bind to both the 2F5 or 4E10 epitope and lipid epitopes. AIDS 2009; 23:2069–2077.
40. Julien JP, Huarte N, Maeso R, Taneva SG, Cunningham A, Nieva JL, Pai EF. Ablation of the complementarity-determining region H3 apex of the anti-HIV-1 broadly neutralizing antibody 2F5 abrogates neutralizing capacity without affecting core epitope binding. J Virol 2010; 84:4136–4147.
41. Pejchal R, Gach JS, Brunel FM, Cardoso RM, Stanfield RL, Dawson PE, et al. A conformational switch in human immunodeficiency virus gp41 revealed by the structures of overlapping epitopes recognized by neutralizing antibodies. J Virol 2009; 83:8451–8462.
42. Scherer EM, Leaman DP, Zwick MB, McMichael AJ, Burton DR. Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions. Proc Natl Acad Sci U S A 2010; 107:1529–1534.
43. Shen X, Dennison SM, Liu P, Gao F, Jaeger F, Montefiori DC, et al.Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution. Proc Natl Acad Sci U S A 2010; 107:5972–5977.
44. Xu H, Song L, Kim M, Holmes MA, Kraft Z, Sellhorn G, et al.Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity. J Virol 2010; 84:1076–1088.
45. Chiantia S, Kahya N, Schwille P. Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances. Langmuir 2005; 21:6317–6323.
46. Chiantia S, Kahya N, Ries J, Schwille P. Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS. Biophys J 2006; 90:4500–4508.
47. Chiantia S, Ries J, Schwille P. Fluorescence correlation spectroscopy in membrane structure elucidation. Biochim Biophys Acta 2009; 1788:225–233.
48. Ries J, Chiantia S, Schwille P. Accurate determination of membrane dynamics with line-scan FCS. Biophys J 2009; 96:1999–2008.
49. Filippov A, Oradd G, Lindblom G. Lipid lateral diffusion in ordered and disordered phases in raft mixtures. Biophys J 2004; 86:891–896.
50. Chiantia S, Ries J, Kahya N, Schwille P. Combined AFM and two-focus SFCS study of raft-exhibiting model membranes. Chemphyschem 2006; 7:2409–2418.
51. Brugger B, Glass B, Haberkant P, Leibrecht I, Wieland FT, Krausslich HG. The HIV lipidome: a raft with an unusual composition. Proc Natl Acad Sci U S A 2006; 103:2641–2646.
52. Moreno MR, Pascual R, Villalain J. Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan. Biochim Biophys Acta 2004; 1661:97–105.
53. Dennison SM, Stewart SM, Stempel KC, Liao HX, Haynes BF, Alam SM. Stable docking of neutralizing human immunodeficiency virus type 1 gp41 membrane-proximal external region monoclonal antibodies 2F5 and 4E10 is dependent on the membrane immersion depth of their epitope regions. J Virol 2009; 83:10211–10223.
54. Song L, Sun ZY, Coleman KE, Zwick MB, Gach JS, Wang JH, et al. Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface. Proc Natl Acad Sci U S A 2009; 106:9057–9062.

2F5; 4E10; gp41; HIV-1; membrane proximal external region; microscopy; supported lipid bilayers

Supplemental Digital Content

© 2011 Lippincott Williams & Wilkins, Inc.